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ObjectiveThe differential expression of microRNAs (miRNAs) between the active stage and the remission stage of ulcerative colitis (UC) was analyzed by bioinformatics method, and the regulatory relationship was constructed by screening the differentially expressed genes (DEGs). The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database (GEO), and the most differentially expressed miRNAs were screened by GEO2R, Excel, and other tools as research objects. TargetScan, miRTarbase, miRDB, STRING, TRRUST, and Matescape databases were used to screen key DEGs, predict downstream transcription factors (TFs), gene ontology (GO), and conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments, the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate (DSS). Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days, and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor (SOCS1), phosphorylated transcriptional signal transductor and activator 3 (p-STAT3), phosphorylated Janus kinase 2 (p-JAK2), and retinoic acid-associated orphan receptor-γt (ROR-γt). The expression levels of transforming growth factor-β (TGF-β), interleukin-17 (IL-17), interleukin-6 (IL-6), and interleukin-10 (IL-10) in serum were detected by enzyme linked immunosorbent assay (ELISA). ResultThe GSE48957 dataset was screened from the GEO database, and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation, as well as signaling pathways such as stem cell signaling pathway, IL-17 signaling pathway, and helper T cell 17 (Th17) cell differentiation. The Matescape database was used to screen out 10 key DEGs, among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3, p-JAK2, and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice, up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10, increase the level of anti-inflammatory factors, and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice, inhibit the phosphorylation of STAT3, inhibit the differentiation of CD4+ T cells into Th17 cells, reduce the levels of pro-inflammatory factors (IL-17 and IL-6), and increase the levels of anti-inflammatory factors (TGF-β and IL-10). As a result, the inflammation of colon mucosa in UC mice was alleviated.
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Objective:Based on the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway,to explore the influences of Liyan syrup on pharyngeal tissue injury and pharyngeal mucosal repair in rats with chronic phar-yngitis.Methods:SD rats were randomly separated into control group,model group,Liyan syrup group,RO8191(JAK2/STAT3 acti-vator)group,Liyan syrup+RO8191 group,the model group and drug intervention group were treated with ammonia water to stimulate the pharynx to build a chronic pharyngitis model,while rats in control group were injected with an equal dose of normal saline into the pharynx.After the intervention of Liyan Syrup and RO8191,the general condition of rats and the apparent state of pharynx were detected,and the apparent state of pharynx was scored;the pathological changes of pharynx of rats were measured by HE staining;the proportions of CD4+T and CD8+T and the ratio of CD4+T/CD8+T in peripheral blood T lymphocyte subsets of rats were measured by flow cytometry;the levels of serum tumor necrosis factor alpha(TNF-α),IL-6,IL-10,malondialdehyde(MDA),reactive oxygen species(ROS)and total antioxidant capacity(T-AOC)were measured by kits;the expressions of JAK2/STAT3 pathway-related proteins in rat pharyngeal tissues were measured by immunoblotting.Results:Compared with control group,the pharyngeal tissue of model group showed obvious pathological morphological damage,the peripheral blood CD4+T proportion and CD4+T/CD8+T,IL-10 and serum T-AOC level decreased(P<0.05),the pharyngeal appearance state score,CD8+T ratio,serum TNF-α,IL-6,MDA and ROS levels,and pharyngeal tissue p-JAK2/JAK2 and p-STAT3/STAT3 levels increased(P<0.05);compared with model group and Liyan syrup+ RO8191 group,the pathological and morphological damage of the pharynx of rats in Liyan syrup group was alleviated,the peripheral blood CD4+T and CD4+T/CD8+T,IL-10,and serum T-AOC level all increased(P<0.05),and the pharyngeal appearance state score,CD8+T,serum TNF-α,IL-6,MDA and ROS levels,and pharyngeal tissue p-JAK2/JAK2 and p-STAT3/STAT3 levels all decreased(P<0.05);the change trend of each index in RO8191 group was opposite to that in Liyan syrup group.Conclusion:Liyan syrup can reduce inflammation and oxidative stress by inhibiting the activation of JAK2/STAT3 signal,enhance anti-inflammatory and antioxidant capacity,relieve throat tissue damage and repair pharyngeal mucosa in rats with chronic pharyngitis.
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Objective:To investigate the effect and mechanism of Grifola frondosa extract on inflammatory response of colon tissue in rats with ulcerative colitis(UC)by regulating interleukin-6(IL-6)/Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods:Forty SD rats were randomly divided into blank control group,UC model group,Grifola frondosa treatment group,western medicine treatment group and combined treatment group,with 8 rats in each group.After UC rats were established by free drinking 3%DSS for 7 days,the treatment group were given Grifola frondosa extract 10 mg/(kg·d),sulfasalazine 0.3 g/(kg·d),and the same amount of two drugs,for 14 consecutive days.During the experiment,general state of rats were observed,and the disease activity index(DAI)score was calculated;pathological changes of rats colon tissue were observed by HE staining;protein expression levels of IL-6,JAK2,STAT3 and p-STAT3 in rats colon tissue were detected by Western blot;content of IL-6 in rats serum was detected by ELISA;protein contents and expressions of IL-6R and MPO in rats colon tissue were determined by immunohistochemistry.Results:Compared with blank control group,general state of rats in UC model group was poor,DAI score was increased,obvious tissue mucosal defects and inflammatory cell infiltration were observed by HE staining;protein expression levels of IL-6,JAK2,STAT3 and p-STAT3 in rats colon tissue and contents of IL-6R and MPO were significantly increased(P<0.01);content of IL-6 in rats serum was significantly increased(P<0.01),the difference was statistically significant.Compared with UC model group,general condition of rats in each treatment group was improved,DAI score was decreased,HE staining showed that mucosal defects were improved to varying degrees,and occasionally inflammatory cell infiltration was observed;protein expression levels of IL-6,JAK2,STAT3 and p-STAT3 in colon tissue were significantly decreased(P<0.01),contents of IL-6R and MPO in colon tissue and content of IL-6 in serum were significantly decreased(P<0.01 or P<0.05),the differences were statistically significant.Conclusion:Grifola frondosa extract can reduce the inflammatory response in colon tissue of UC rats by regulating expressions of IL-6/JAK2/STAT3 signaling pathway related factors.
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ObjectiveTo observe the effect of Youguiwan on the leptin/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in the lung tissue of the rat model of chronic obstructive pulmonary disease (COPD) due to kidney-Yang deficiency. MethodForty rats were modeled for COPD with the syndrome of kidney-Yang deficiency by intratracheal instillation of lipopolysaccharide on day 1 and day 14 and continuous fumigation for 6 weeks, during which hydrocortisone was injected intramuscularly at an interval of 3 days. The modeled rats were randomized into model, high- (11.7 g·kg-1), medium- (5.85 g·kg-1), and low-dose (2.93 g·kg-1) Youguiwan, and aminophylline (0.054 g·kg-1) group. In addition, 8 SD rats were set as the blank group. After the completion of modeling, the rats in each group were administrated with the corresponding drug by gavage for 28 consecutive days. After the last administration, samples were collected. A lung function analyzer was used to evaluate the lung function of rats. Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin-17A (IL-17A), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bronchoalveolar lavage fluid (BALF). Hematoxylin-eosin staining was employed to observe the pathological changes in the lung tissue, and Masson staining was employed to observe the deposition of blue collagen fibers around bronchi in the lung tissue and calculate the inflammation score. The immunofluorescence assay was employed to measure the protein content of collagen type Ⅰ (ColⅠ) and α-smooth muscle actin (α-SMA) in the bronchi. The protein and mRNA levels of leptin, IL-17A, JAK2, and STAT3 in the lung tissue were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction, respectively. ResultCompared with the blank group, the model group showed decreased lung function (P<0.01), elevated levels of IL-6, IL-17A, and TNF-α in the BALF (P<0.01), and increased lung inflammation score, deposition of subcutaneous collagen fibers in the airway, and ColⅠ and α-SMA proteins (P<0.01). Furthermore, the modeling up-regulated the proteins and mRNA levels of leptin, IL-17A, JAK2, and STAT3 in the lung tissue (P<0.01) and enhanced the phosphorylation of JAK2 and STAT3 (P<0.01). Compared with the model group, high- and medium-dose Youguiwan improved the lung function, decreased the inflammation score, reduced collagen fiber deposition and ColⅠ and α-SMA proteins, lowered the levels of IL-6, IL-17A, and TNF-α in the BALF, down-regulated the mRNA and protein levels of leptin, JAK2, STAT3, and IL-17A, and weakened the phosphorylation of JAK2 and STAT3 (P<0.05, P<0.01). The aminophylline group had higher IL-17A and TNF-α levels than the high-dose Youguiwan group, lower IL-17A level than the medium and low-dose Youguiwan groups, and lower TNF-α level than the low-dose Youguiwan group. Compared with the aminophylline group, the high- and medium-dose Youguiwan groups showed reduced deposition of collagen fibers and protein levels of ColⅠ and α-SMA around the bronchi in the lung tissue (P<0.05, P<0.01), decreased inflammation score, and down-regulated protein and mRNA levels of leptin, JAK2, STAT3, and IL-17A in the lung tissue. ConclusionYouguiwan can prevent airway remodeling by inhibiting IL-17A to reduce inflammation and collagen deposition in COPD rats, which may be related to the inhibition of the leptin/JAK2/STAT3 signaling pathway.
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@#Abstract: Objective To investigate the influences of notoginsenoside R1 (NGR1) on cell injury and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway of alveolar epithelial cells infected by Klebsiella pneumoniae (Kp). Methods A549 cells were grouped into five groups: control group (C group), infection group (Infect group), infection + low NGR1 group (Infect + L-NGR1 group), infection + high NGR1 group (Infect + H-NGR1 group), and infection+high NGR1+JAK2/STAT3 pathway inhibitor group (Infect+H-NGR1+SD-1029 group). Cell proliferation was measured using CCK8; ELISA kits were applied to detect the contents of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon γ (IFN-γ) in the culture medium; flow cytometry was applied to detect apoptosis; RT-qPCR was applied to detect the expressions of JAK2/STAT3; Western blot was applied to detect JAK2/STAT3 pathway, autophagy protein microtubule-associated protein 1 light chain 3 (LC3), autophagy-relatedgene5 (Atg5), autophagy-related gene (Atg) 6 (Beclin-1), apoptosis protein B-cell lymphoma 2 (Bcl-2), Bcl-2-accociated protein (Bax), cysteinyl aspartate specific proteinase (cleaved-caspase-3) proteins expression. Results Compared with the C group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the mRNA relative expressions and protein phosphorylation levels of JAK2, STAT3 in the Infect group were obviously decreased (P<0.05); the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-caspase-3 were obviously increased (P<0.05). Compared with Infect group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the mRNA relative expressions and protein phosphorylation levels of JAK2, STAT3 in the Infect+L-NGR1 group and Infect+H-NGR1 group were obviously increased (P<0.05); the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-Caspase-3 were obviously decreased (P<0.05). Compared with Infect+H-NGR1 group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the protein phosphorylation levels of JAK2, STAT3 in the Infect+H-NGR1+SD-1029 group were obviously decreased (P<0.05), and the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-caspase-3 were obviously increased (P<0.05). Conclusions NGR1 can activate the JAK2/STAT3 signaling pathway, promote autophagy of alveolar epithelial cells, and inhibit Kp-induced inflammatory injury and apoptosis of alveolar epithelial cells.
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Objective:To investigate the effects of mild hypothermia on microglia polarization and janus kinase 2/signal transduction and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway during cerebral ischemia-reperfusion (I/R) in rats.Methods:Forty-five clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 3 groups ( n=15 each) by the random number table method: sham operation group (S group), cerebral I/R group (I/R) and mild hypothermia group (H group). In I/R group and H group, cerebral I/R was induced by middle cerebral artery occlusion using a nylon thread in anesthetized animals, the nylon thread was removed to restore the perfusion after 2 h of occlusion, and the rectal temperature was maintained at 36-37 ℃ during the period. Group H was wiped with 75% alcohol for 3 h starting from the time point immediately after reperfusion, and the rectal temperature was maintained at 32-33℃. Modified neurological severity score (mNSS) was evaluated at 24 h of reperfusion. Animals were then sacrificed for determination of the cerebral infarct size (using TTC staining), expression of M1 marker inducible nitric oxide synthase (iNOS), M2 marker arginase 1(Arg-1), phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)(by Western blot), expression of iNOS mRNA and Arg-1 mRNA (by quantitative polymerase chain reaction), and contents of interleukin-6 (IL-6) and IL-10 (by enzyme-linked immunosorbent assay). Results:Compared with group S, mNSS and cerebral infarct size were significantly increased, the expression of iNOS, Arg-1 protein and mRNA in cerebral ischemic penumbral zone was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio, and contents of IL-6 and IL-10 were increased in the other two groups ( P<0.05). Compared with I/R group, mNSS and cerebral infarct size were significantly decreased, the expression of iNOS protein and mRNA in cerebral ischemic penumbral zone was down-regulated, the expression of Arg-1 and mRNA was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio and IL-6 content were decreased, and the IL-10 content was increased in group H ( P<0.05). Conclusions:Mild hypothermia can promote the polarization shift of microglia from M1 to M2 phenotype during cerebral I/R and inhibit the central inflammatory responses, and the mechanism may be related to inhibition of JAK2/STAT3 signaling pathway in rats.
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ObjectiveTo investigate the effect of Qiling Baitouweng Tang (QLBTWT) on proliferation and apoptosis, Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway and interleukin-10 (IL-10) in diffuse large B-cell lymphoma (DLBCL). MethodWith human DLBCL cells OCI-LY10 and U2932 as research objects, cell proliferation was detected by cell counting kit-8 (CCK-8) assay. After treatment with 0, 4.6, 9.3, 18.7, 37.5, 75, 150 mg·L-1 QLBTWT for 24 h, the half-inhibitory concentration (IC50) of OCL-LY10 and U2932 cells was calculated to be 9.33, 16.13 mg·L-1, respectively, based on which, 9.5, 19, 38 mg·L-1 QLBTWT were selected for subsequent experiments. After 0, 9.5, 19, 38 mg·L-1 QLBTWT treatment for 24 h, the zymogen activities of Caspase-3, Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits (colorimetric), and the IL-10 expression was detected by enzyme-linked immuno sorbent assay (ELISA). The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved poly adenosine diphosphate ribose polymerase (cleaved PARP), cleaved Caspase-3], JAK2, STAT3, phospho-JAK2 (p-JAK2), phospho-STAT3 (p-STAT3) pathway proteins, and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0, 9.5, 19, 38 mg·L-1 QLBTWT were all tested by Western blot. ResultAfter QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h, cell proliferation was inhibited in each QLBTWT group compared with that in the control group (P<0.05, P<0.01). The zymogens of Caspase-3, Caspase-8 and Caspase-9 were activated (P<0.01), and there was an increase in cell apoptosis (P<0.05, P<0.01) and cell cycle arrest at Gap phase1 (G1) phase in 9.5, 19 and 38 mg·L-1 QLBTWT group (P<0.05, P<0.01). After 9.5, 19 and 38 mg·L-1 QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h, the expressions of Bcl-2, p-JAK2 and p-STAT3 proteins were decreased (P<0.01), and the expressions of Bax, cleaved PARP and cleaved Caspase-3 proteins were increased (P<0.01), but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group, the expressions of c-Myc, p-JAK2, and p-STAT3 proteins were down-regulated in 19 mg·L-1 QLBTWT group and 19 mg·L-1 QLBTWT+10 μg·L-1 IL-10 group (P<0.05, P<0.01), and up-regulated in 10 μg·L-1 IL-10 group (P<0.05, P<0.01), while there was no difference in JAK2/STAT3 proteins. ConclusionQLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932, and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.
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ObjectiveTo observe the effect of Hedysari Radix polysaccharide (HRP) on the Janus kinase 2 (JAK2)/signal transducer and activator of transcription protein 3 (STAT3) signaling pathway in diabetic nephropathy db/db mice. MethodFifty db/db mice were randomly divided into model group, irbesartan group (irbesartan suspension, 22.75 mg·kg-1), and high-, medium-, and low-dose HRP groups (HRP suspension, 200, 100, 50 mg·kg-1) according to the body weight, with 10 mice in each group. Another 10 C57BL/6 mice were assigned to the normal group. The mice were treated with corresponding drugs by gavage, while those in the normal group and the model group received distilled water at 5 mL·kg-1. The mice in the six groups were administered once a day by gavage for 12 consecutive weeks. The uric acid (UA), triglycerides (TG), and total cholesterol (TC) were detected. Periodic acid-Schiff (PAS) staining and Masson staining were used to observe the pathological changes in kidney tissues. Western blot and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) were used to detect the protein and mRNA expression levels of JAK2, STAT3, suppressor of cytokine signaling 3 (SOCS3), and tumor necrosis factor-α (TNF-α) in the kidney. ResultAfter 12 weeks of treatment, compared with the normal group, the model group showed significant pathological ultrastructural changes in kidney tissues and increased UA, TG, and TC levels (P<0.01). Compared with the model group, the high- and medium-dose HRP groups and the irbesartan group showed improvement in pathological ultrastructure of kidney tissues and reduced UA, TG, and TC levels (P<0.05, P<0.01). Compared with the normal group, the model group showed a decrease in SOCS3 protein and mRNA expression levels and an increase in JAK2, STAT3, and TNF-α protein and mRNA expression levels (P<0.01). Compared with the model group, the high- and medium-dose HRP groups and the irbesartan group showed an increase in SOCS3 protein and mRNA expression levels and a decrease in JAK2, STAT3, and TNF-α protein and mRNA expression levels (P<0.05, P<0.01). ConclusionHRP can alleviate renal damage in diabetic nephropathy to a certain extent, and its mechanism may be related to the inhibition of the activation of the JAK2/STAT3 signaling pathway.
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Objective @# To investigate the effect of pathogenic bacterium-Porphyromonas gingivalis (P.g) on the proliferation and inflammatory factor expression of human colorectal cancer Caco-2 cells, to determine whether the Janus kinase 2-signal transducers and activators of transcription 3 (JAK2-STAT3) pathway is involved in the regulation of Caco-2 cell proliferation by P.g and to provide an experimental basis for further exploring the relationship between P.g and colorectal cancer. @*Methods @# Caco-2 cells were cultured in vitro, and P.g at different multiplicities of infection (MOIs) (0, 1, 10, 25) was selected to stimulate for 12, 24 and 48 h. The effect of P.g on the proliferation of Caco-2 cells was detected by CCK8. The stimulation time was set as 12, 24 and 48 h. MOI=0 was the control group, and MOI=1, 10 and 25 comprised the experimental group. qRT-PCR and Western blot were used to detect the changes in interleukin-6 (IL-6), interleukin-10(IL-10), JAK2 and STAT3 gene and protein (phosphorylated protein) levels in each group. @*Results @# After P.g infection of Caco-2 cells, P.g had a sustained stimulatory effect on the cells for 12, 24 and 48 h at MOI=1 and MOI=10 compared with the control group. Compared with that in the control group, the expression of pro-inflammatory factor IL-6 and related proliferative pathway protein JAK2 and STAT3 in Caco-2 cells with P.g infection increased in a concentration- and time-dependent manner (P<0.05). Additionally, the expression of IL-10, an anti-inflammatory factor, in Caco-2 cells infected with P.g decreased (P<0.05). After the addition of the JAK2 inhibitor AZ960, the proliferation of Caco-2 cells infected with P.g decreased, and the mRNA expression of STAT3 and JAK2 and the protein expression of p-STAT3 and p-JAK2 decreased (P<0.05). @*Conclusion @#P.g can promote the proliferation of the colorectal cancer cell line Caco-2, and the effect of P.g on Caco-2 cells may promote cell proliferation through the JAK2-STAT3 pathway while promoting the expression of the proinflammatory factor IL-6 and inhibiting the expression of the anti-inflammatory factor IL-10, creating an inflammatory environment conducive to cell proliferation, which may be the mechanism by which P.g affects the proliferation of Caco-2 cells.
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Interleukin-6 (IL-6) is a spreading pleiotropic cytokine, with both anti-inflammatory and proinflammatory effects. It not only participates in the body immune responses but also is involved in the biological regulative processes among different organs, tissues, and cells. IL-6 has both anti-inflammatory and pro-inflammatory effects. In the early stage of pathogen infection, IL-6 plays an anti-inflammatory role in the body, and its level is moderately increased in the body to resist inflammation and maintain internal homeostasis. However, a large amount of IL-6 release can cause excessive inflammation and trigger other pathological changes in the body. Il-6 also has the dual effect of stimulating the synthesis and degradation of skeletal muscle protein in regulating skeletal muscle mass. As an important locomotive organ, skeletal muscle is also one of the key targets of IL-6. IL-6 takes part in the biological control of skeletal muscle hypertrophy through regulating muscle satellite cell proliferation and differentiation under specific stresses. In addition IL-6 is also associated with skeletal muscle atrophy induced by aging and other pathological stresses. In addition, during exercise stress, skeletal muscle can also serve as an endocrine organ to secrete and release IL-6 that facilitates the "crosstalk" between skeletal muscle and other organs or tissues. As IL-6 plays as a versatile role in our body, this paper reviews the research progress of the mechanism of IL-6 in the regulation of skeletal muscle mass, which may provide theoretical support for revealing the molecular mechanism of skeletal muscle stresses and adaptations.
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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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Female , Humans , Janus Kinase 2 , Caspase 3 , bcl-2-Associated X Protein , Interleukin-6/genetics , Apoptosis , Signal Transduction , Cell Proliferation , STAT3 Transcription Factor/geneticsABSTRACT
OBJECTIVE@#To investigate the effect of kaempferol on proliferation of acute myeloid leukemia (AML) KG1a cells and its mechanism.@*METHODS@#Human AML KG1a cells in logarithmic growth stage were taken and set at 25, 50, 75 and 100 μg/ml kaempferol group, another normal control group (complete medium without drug) and solvent control group (add dimethyl sulfoxide) were also set. After 24 and 48 hours of intervention, the cell proliferation rate was detected by CCK-8 assay. In addition, interleukin-6 (IL-6) combined with kaempferol group (Plus 20 μg/l IL-6 and 75 μg/ml kaempferol) was set up, 48 hours after culture, the cell cycle and apoptosis of KG1a cells were detected by flow cytometry, the mitochondrial membrane potential (MMP) of KG1a cells was detected by MMP detection kit (JC-1 method), and the expression of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway related proteins in KG1a cells were detected by Western blot.@*RESULTS@#The cell proliferation rate of 25, 50, 75 and 100 μg/ml kaempferol group decreased significantly (P<0.05), and with the increase of kaempferol dose (r24 h=-0.990, r48 h= -0.999), the cell proliferation rate decreased gradually (P<0.05). The inhibitory effect of 75 μg/ml kaempferol on cell proliferation reached half of effective dose after 48 hours of intervention. Compared with normal control group, the G0/G1 phase cell proportion and apoptosis rate of cells in 25, 50 and 75 μg/ml kaempferol group increased, while the S phase cell proportion, MMP, phosphorylated JAK2 (p-JAK2)/JAK2 and phosphorylated STAT3 (p-STAT3)/STAT3 protein expression decreased in a dose-dependent manner (r=0.998, 0.994, -0.996, -0.981, -0.997, -0.930). Compared with 75 μg/ml kaempferol group, the G0/G1 phase cell proportion and apoptosis rate of cells in IL-6 combined with kaempferol group decreased, while the S phase cell proportion, MMP, p-JAK2/JAK2 and p-STAT3/STAT3 protein expression increased significantly (P<0.05).@*CONCLUSION@#Kaempferol can inhibit KG1a cell proliferation and induce KG1a cell apoptosis, its mechanism may be related to the inhibition of JAK2/STAT3 signal pathway.
Subject(s)
Humans , STAT3 Transcription Factor/metabolism , Interleukin-6/metabolism , Kaempferols/pharmacology , Signal Transduction , Apoptosis , Janus Kinase 2 , Cell Proliferation , Leukemia, Myeloid, AcuteABSTRACT
BACKGROUND: Philadelphia negative myeloproliferative neoplasms (Ph-MPN) are clonal disorders whose pathogenesis has been elucidated in recent years, creating diagnostic and prognostic algorithms. AIM: To study JAK2, CALR y MPL gene mutations in patients with Ph-MPN. MATERIALS AND METHODS: Descriptive cross-sectional observational study of patients with MPN (2015-2019), reviewing clinical, demographic and laboratory data. JAK2, CALR and MPL gene mutations were analyzed by RT-PCR. Results: We studied 72 patients. Fifty percent had essential thrombocythemia (ET), 26.4% had polycythemia vera (PV) and 23.6% had primary myelofibrosis (PM). Bone marrow biopsy was available in 76.5%. At diagnosis, the mean age was 65.5 years and 61% were symptomatic. A thrombotic event was the most frequent problem in 20% and 25% had splenomegaly. There were statistically significant differences in hematological parameters between the different MPNs. JAK2 V617F mutation was detected in 61.1%. Only 19 JAK2 V617F negative patients were available for CALR and MPL mutation studies, identifying 10 triple negative cases. Kaplan Meier curves showed a median survival of 88 months, being similar in the three MPNs. Causes of death in 20 patients were thrombotic complications in 30%, disease progression in 25%, infection in 20%, other neoplasms in 15% and other causes in 10%. CONCLUSIONS: The presentation and frequency of JAK2 V617F, CALR and MPL mutations in our cohort was similar to those reported in other studies for ET and PM. JAK2 V617F mutation was lower for PV. No significant differences between the three MPNs were observed for overall survival. We could not assess the prognostic value of the mutations.
Subject(s)
Humans , Aged, 80 and over , Polycythemia Vera/genetics , Thrombocythemia, Essential/diagnosis , Thrombocythemia, Essential/genetics , Myeloproliferative Disorders/genetics , Chile , Cross-Sectional Studies , Hospitals, Public , MutationABSTRACT
Objective @#To investigate the effect of Rotundic acid (RA) on proliferation,migration and invasion a- bility of human lung adenocarcinoma cells as well as its possible mechanisms.@*Methods @#Human lung adenocarci- noma A549 and PC9 cells were divided into control group,blank control group,solvent group and 20,40,60,80 μmol / L RA groups.CCK-8 assay and scratch assay were used to detect the proliferation and horizontal migration of human lung adenocarcinoma cells.Transwell migration assay and Transwell invasion assay were used to detect the longitudinal migration and invasion ability of A549 and PC9 cells in each group.The protein expression levels of ja- nus kinase 2 (JAK2) and signal transducer and activator of transcription 3 ( STAT3) in the supernatants of A549 and PC9 cells were detected by ELISA.The mRNA expression levels of JAK2 and STAT3 were detected by RT- PCR. Statistical analysis was made on the differences among groups in each index. @*Results @#After RA treatment on human lung adenocarcinoma cells ,compared with the control group ,the proliferation activity of A549 and PC9 cells in the experimental groups decreased (P<0. 05) ,the number of cells crossing polycarbonate membrane and matrix glue decreased (P<0. 05) ,the expression of JAK2 and STAT3 proteins in cell supernatant decreased (P < 0. 05) ,and the mRNA expression of JAK2 and STAT3 decreased (P<0. 05) .The decrease of the above indices was concentration-dependent and had statistical significance (P<0. 05) .Compared with the control group,the pro- liferation activity of A549 and PC9 cells in the solvent group showed no significant difference.@*Conclusion @#RA may inhibit the proliferation,migration and invasion of human lung adenocarcinoma A549 and PC9 cells in vitro, possibly through the inhibition of JAK2 / STAT3 pathway.
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@#BACKGROUND: Exosomes and exosomal microRNAs have been implicated in tumor occurrence and metastasis. Our previous study showed that microRNA-761 (miR-761) is overexpressed in hepatocellular carcinoma (HCC) tissues and that its inhibition affects mitochondrial function and inhibits HCC metastasis. The mechanism by which exosomal miR-761 modulates the tumor microenvironment has not been elucidated. METHODS: Exosomal miR-761 was detected in six cell lines. Cell counting kit-8 (CCK-8) and transwell migration assays were performed to determine the function of exosomal miR-761 in HCC cells. The luciferase reporter assay was used to analyze miR-761 target genes in normal fibroblasts (NFs). The inhibitors AZD1480 and C188-9 were employed to determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in the transformation of cancer-associated fibroblasts (CAFs). RESULTS: In this study, we characterized the mechanism by which miR-761 reprogrammed the tumor microenvironment. We found that HCC-derived exosomal miR-761 was taken up by NFs. Moreover, HCC exosomes affected the tumor microenvironment by activating NFs via suppressor of cytokine signaling 2 (SOCS2) and the JAK2/STAT3 signaling pathway. CONCLUSIONS: These results demonstrated that exosomal miR-761 modulated the tumor microenvironment via SOCS2/JAK2/STAT3 pathway-dependent activation of CAFs. Our findings may inspire new strategies for HCC prevention and therapy.
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ObjectiveTo explore the mechanism of Xueniao capsule in the treatment of acute pyelonephritis (APN) by network pharmacology and experimental verification. MethodThe effect of Xueniao capsule on APN was investigated based on the APN model in rats. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Chemistryl Database, and SymMap were searched for the chemical components of Smilacis Chinae Rhizoma,Coicis Semen, and Trachycarpi Petiolus. The target information of the components was collected from PharmMapper and SwissTargetPrediction, and disease target information from Therapeutic Target Database (TTD), DrugBank, DisGeNET, GeneCards, and Online Mendelian Inheritance in Man(OMIM). The key genes of Xueniao capsule for APN underwent Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses by Metascap. Real-time quantitative polymerase chain reaction (PCR) and Western blot were employed to verify the prediction results. ResultCompared with the blank group and the sham operation group, the model group showed an increased ratio of the left kidney to the right kidney and organ index(P<0.05, P<0.01),up-regulated white blood cells (WBC),neutrophils (NEUT),monocytes (MONO), and lymphocytes (LY)(P<0.05, P<0.01), and elevated levels of nuclear factor-κB(NF-κB), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)(P<0.05, P<0.01). Compared with the model group, the norfloxacin group, the low- and high-dose Xueniao capsule groups showed a decreased ratio of the left kidney to the right kidney and organ index(P<0.05, P<0.01), dwindled levels of WBC, NEUT, MONO, and LY(P<0.05, P<0.01), and reduced levels of NF-κB, IL-6, and TNF-α(P<0.05, P<0.01). The medium-dose Xueniao capsule group showed a decreased ratio of the left kidney to the right kidney and organ index(P<0.05, P<0.01), reduced levels of WBC, NEUT, MONO, and LY(P<0.05, P<0.01), and dwindled levels of IL-6 and TNF-α(P<0.05, P<0.01). Network pharmacological analysis revealed 17 active compounds from Smilacis Chinae Rhizoma, 18 active compounds from Coicis Semen, six active compounds from Trachycarpi Petiolus, and 39 key genes for the treatment of APN in Xueniao capsule. GO enrichment analysis demonstrated 704 biological processes, 22 cellular components, and 59 molecular functions. Sixty-two pathways were enriched in KEGG enrichment analysis. The experimental verification results showed that compared with the blank group, the model group showed increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), mitogen-activated protein kinase 1 (MAPK1)/extracellular signal-regulated protein kinase 2 (ERK2),phosphoinositide 3 kinase (PI3K),protein kinase B2(Akt2),Janus kinase 2 (JAK2),and signal transducer and activator of transcription 3 (STAT3)and protein expression of PI3K, Akt2, JAK2, and STAT3 (P<0.05, P<0.01). Compared with the model group, the low-dose Xueniao capsule group showed decreased mRNA expression of MAPK1, PI3K, JAK2, and STAT3 and protein expression of PI3K, JAK2, and STAT3 (P<0.05, P<0.01). The medium-dose Xueniao capsule group showed decreased mRNA expression of MAPK1, PTGS2, PI3K, JAK2, and STAT3, and protein expression of PI3K, JAK2, and STAT3 (P<0.05, P<0.01). The high-dose Xueniao capsule group showed reduced mRNA expression of PTGS2, MAPK1, PI3K, Akt2, JAK2, and STAT3 and protein expression of PI3K, Akt2, JAK2, and STAT3 (P<0.05, P<0.01). ConclusionXueniao capsule has a certain curative effect on APN via multiple targets and multiple pathways. The mechanism may be related to the inhibition of the PI3K/Akt signaling pathway and the JAK2/STAT3 signaling pathway.
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ObjectiveTo investigate the effect of pulsatilla saponin A (PSA) on proliferation and apoptosis of human Burkitt lymphoma (BL) cell line Raji cells and expression of related pathway proteins. MethodWith Raji cells as the research object, the cell proliferation was detected by cell counting kit-8 (CCK-8) method, and the half-maximal inhibitory concentration (IC50) values of 24 h, 48 h and 72 h were calculated to be 19.77, 18.31, 16.70 μmol·L-1, respectively. In subsequent related experiments, 0, 8, 16, 32 μmol·L-1 PSA were selected according to the IC50 value of Raji cells treated with PAS for 72 h. After 0, 8, 16, 32 μmol·L-1 PSA acted on Raji cells for 24, 48, 72 h, the optical density values of cell growth curve were detected by CCK-8 method. The zymogen activities of cysteine aspartate-specific protease (Caspase)-3, Caspase-8 and Caspase-9 in Raji cells treated with 0, 8, 16 and 32 μmol·L-1 PSA for 24 h were measured by Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kit. The apoptosis rate and cell cycle of Raji cells treated with different concentrations of PSA after 24 h were detected by flow cytometry. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved poly(ADP-ribose) polymerase (cleaved PARP), cleaved cysteinyl aspartate-specific protease-3 (cleaved Caspase-3) apoptosis related protein and Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phosphorylated-JAK2 (p-JAK2), and phosphorylated- STAT3 (p-STAT3) pathway proteins in Raji cells after 24 h of treatment with 0, 8, 16 and 32 μmol·L-1 PSA were tested by Western blot. ResultCompared with control group, decreased cell survival rate, inhibited cell proliferation, activated zymogens of Caspase-3, Caspase-8 and Caspase-9 (P<0.01), increased apoptosis (P<0.05, P<0.01), and enhanced cell cycle arrest in Gap phase 2 (G2) were observed in 8, 16 and 32 μmol·L-1 PSA groups(P<0.05, P<0.01). Compared with control group, cells treated with 8, 16 and 32 μmol·L-1 PSA had lower expression of Bcl-2, p-JAK2, p-STAT3 proteins (P<0.05, P<0.01), and higher expression of Bax, cleaved PARP and cleaved Caspase-3 protein (P<0.01), while no significant change was found in the expression of JAK2 and STAT3 proteins. ConclusionPSA could inhibit proliferation and induce apoptosis of Raji cells, and its potential mechanism might be related to the regulation of JAK2/STAT3 signaling pathway.
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OBJECTIVE@#This study aimed to investigate the effects of caprylic acid (C8:0) on lipid metabolism and inflammation, and examine the mechanisms underlying these effects in mice and cells.@*METHODS@#Fifty-six 6-week-old male C57BL/6J mice were randomly allocated to four groups fed a high-fat diet (HFD) without or with 2% C8:0, palmitic acid (C16:0) or eicosapentaenoic acid (EPA). RAW246.7 cells were randomly divided into five groups: normal, lipopolysaccharide (LPS), LPS+C8:0, LPS+EPA and LPS+cAMP. The serum lipid profiles, inflammatory biomolecules, and ABCA1 and JAK2/STAT3 mRNA and protein expression were measured.@*RESULTS@#C8:0 decreased TC and LDL-C, and increased the HDL-C/LDL-C ratio after injection of LPS. Without LPS, it decreased TC in mice ( P < 0.05). Moreover, C8:0 decreased the inflammatory response after LPS treatment in both mice and cells ( P < 0.05). Mechanistic investigations in C57BL/6J mouse aortas after injection of LPS indicated that C8:0 resulted in higher ABCA1 and JAK2/STAT3 expression than that with HFD, C16:0 and EPA, and resulted in lower TNF-α, NF-κB mRNA expression than that with HFD ( P < 0.05). In RAW 264.7 cells, C8:0 resulted in lower expression of pNF-κBP65 than that in the LPS group, and higher protein expression of ABCA1, p-JAK2 and p-STAT3 than that in the LPS and LPS+cAMP groups ( P < 0.05).@*CONCLUSION@#Our studies demonstrated that C8:0 may play an important role in lipid metabolism and the inflammatory response, and the mechanism may be associated with ABCA1 and the p-JAK2/p-STAT3 signaling pathway.
Subject(s)
Animals , Humans , Male , Mice , ATP Binding Cassette Transporter 1/immunology , Caprylates/chemistry , Cholesterol/metabolism , Diet, High-Fat/adverse effects , Inflammation/metabolism , Janus Kinase 2/immunology , Lipid Metabolism/drug effects , Macrophages/immunology , Mice, Inbred C57BL , STAT3 Transcription Factor/immunology , Signal TransductionABSTRACT
Objective To investigate the protective effect of micro RNA(miR)-98-5p targeting Kruppel-like factor 9(KLF9) against myocardial isehemia-reperfusion(MI/R) injury in rats. Methods Totally 50 rats were randomly divided into sham operation group, model group, miR-98-5p agomir group, agomir-NC group, and miR-98-5p agomir+pcDNA-3. 1-KLF9 group, 10 in each group. MI/R model was established by coronary artery ligation. The pathological changes of myocardial tissue were detected by HE staining. The myocardial apoptosis were detected by TUNEL. The levels of creatine kinase (CK), creatine kinase isoenzymes (CK-MB) and lactate dehydrogenase (LDH) in serum were detected by ELISA. The expression levels of miR-98-5p and KLF9 mRNA were detected by Real-time PCR. The expression of KLF9, Bax and JAK2/STAT3 pathway relative protein were detected by Western blotting. Dual luciferase assay verified the relationship between miR-98-5p and KLF9. Results Compared with the sham operation group, the arrangement of myocardial cells in the model group was disordered, and the myocardial cells appeared necrosis. The apoptosis rate of myocardial cells, serum CK, CK-MB and LDH contents increased, the expression level of miR-98-5p decreased, and the expression levels of KLF9 mRNA and protein, p-JAK2 and p-STAT3 protein increased (P < 0. 05) . After the overexpression of miR-98-5p, the myocardial cells arranged more orderly and the myocardial cell necrosis decreased. The apoptosis rate of myocardial tissue, the contents of CK, CK-MB and LDH in serum and the expression of p-JAK2 and p-STAT3 protein were decreased (P< 0. 05) . The result of dual luciferase assay showed that KLF9 was the target gene of miR-98-5p. The overexpression of KLF9 reversed the effects of miR-98-5p agomir on myocardial injury. Conclusion MiR-98-5p targeting KLF9 can improve the myocardial injury of MI/R rats. The mechanism may be related to the regulation of JAK2/STAT3 signal pathway by miR-98-5p which inhibit myocardial cell apoptosis.
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BACKGROUND: Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are chronic hematological disorders characterized by the overproduction of one or more mature myeloid blood cell lineages. Classical Ph-MPN are polycythemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). AIM: To assess the epidemiological, clinical and diagnostic characteristics of Ph-MPN in Chile. Material and Methods: Retrospective review of medical records of all patients referred as MPN from 2012 to 2017. Patients with (9;21) translocation were excluded. RESULTS: Data of 462 cases with a median age of 69 years from 10 public hospitals was reviewed. ET was the most frequently Ph-MNP found. The incidence of Ph-MPN was 1.5 x 100.000 cases. The JAK2 V617F mutation study was performed in 96% of patients and only 30% had a bone marrow biopsy. Thrombotic events were observed in 29% of patients. Bleeding events were observed in 7%. Five-year overall survival was 87%. Conclusions: ET is the most frequent Ph-MPN. The mean incidence was lower than reported in the literature, in part because of a sub diagnosis.