Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

STAT3-Dependent Effects of Polymeric Immunoglobulin Receptor in Regulating Interleukin-17 Signaling and Preventing Autoimmune Hepatitis

One-third of patients with autoimmune hepatitis(AIH)have cirrhosis at the time of diagnosis.The relevance of these variables,although unknown,is believed to be critical in AIH because of suspected interactions between the gut microbiome and genetic factors.Dysbiosis of the gut flora and elevated polymeric immunoglobulin receptor(pIgR)levels have been observed in both patients and mouse models.Moreover,there is a direct relationship between pIgR expression and transaminase levels in patients with AIH.In this study,we aimed to explore how pIgR influences the secretion of regenerating islet-derived 3 beta(Reg3b)and the flora composition in AIH using in vivo experiments involving patients with AIH and a concanavalin A-induced mouse model of AIH.Reg3b expression was reduced in pIgR gene(Pigr)-knockout mice compared to that in wild-type mice,leading to increased microbiota disruption.Conversely,exogenous pIgR supplementation increased Reg3b expression and maintained microbiota homeostasis.RNA sequencing revealed the participation of the interleukin(IL)-17 signaling pathway in the regulation of Reg3b through pIgR.Furthermore,the introduction of external pIgR could not restore the imbalance in gut microbiota in AIH,and the decrease in Reg3b expression was not apparent following the inhibition of signal transducer and activator of transcription 3(STAT3).In this study,pIgR facilitated the upregulation of Reg3b via the STAT3 pathway,which plays a crucial role in preserving the balance of the intestinal microbiota in AIH.Through this research,we discovered new molecular targets that can be used for the diagnosis and treatment of AIH.

Regulation and function of signal transducer and activator of transcription 3

Signal transducer and activator of transcription 3(STAT3), a member of the STAT family, is a key regulator of many physiological and pathological processes. Significant progress has been made in understanding the transcriptional control, posttranslational modification, cellular localization and functional regulation of STAT3. STAT3 can translocate into the nucleus and bind to specific promoter sequences, thereby exerting transcriptional regulation. Recent studies have shown that STAT3 can also translocate into mitochondria, participating in aerobic respiration and apoptosis. In addition, STAT3 plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. Conditional knockout mouse models make it possible to study the physiological function of STAT3 in specific tissues and organs. This review summarizes the latest advances in the understanding of the expression, regulation and function of STAT3 in physiological and tumorigenic processes.

Impact of STAT-signaling pathway on cancer-associated fibroblasts in colorectal cancer and its role in immunosuppression

Colorectal cancer(CRC)remains one of the most commonly diagnosed and deadliest types of cancer worldwide.CRC displays a desmoplastic reaction(DR)that has been inversely associated with poor prognosis;less DR is associated with a better prognosis.This reaction generates excessive connective tissue,in which cancer-associated fibroblasts(CAFs)are critical cells that form a part of the tumor microenvironment.CAFs are directly involved in tumorigenesis through different mechanisms.However,their role in immunosuppression in CRC is not well understood,and the precise role of signal transducers and activators of transcription(STATs)in mediating CAF activity in CRC remains unclear.Among the myriad chemical and biological factors that affect CAFs,different cytokines mediate their function by activating STAT signaling pathways.Thus,the harmful effects of CAFs in favoring tumor growth and invasion may be modulated using STAT inhibitors.Here,we analyze the impact of different STATs on CAF activity and their immunoregulatory role.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis. Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals. After 4 weeks, OVA atomization and gavage were used for stimulation and treatment. Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74, 3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline. HE, PAS and Masson staining were used to observe the lung histopathological changes in rats. The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA. The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue. Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious. The expressions of IL-6, IL- 23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01);Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased (P< 0.01). The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01). Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

Downregulation of signal transducer and activator of transcription 3 by sorafenib:A novel mechanism for hepatocellular carcinoma therapy

Hepatocellular carcinoma is one of the most common cancers worldwide,and a leading cause of cancer-related death.Owing to unsatisfactory clinical outcomes under the current standard of care,there is a need to search for and identify novel and potent therapeutic targets to improve patient outcomes.Sorafenib is the first and only approved targeted therapy for the treatment of hepatocellular carcinoma.Besides functioning as a multiple tyrosine kinase,sorafenib also acts via a kinase-independent mechanism to target signal transducer and activator of transcription 3(STAT3) signaling in hepatocellular carcinoma cells.STAT3 is a key regulator of inflammation,cell survival,and tumorigenesis of liver cells,and the high percentage of hepatocellular carcinoma cells with constitutively active STAT3 justifies targeting it for the development of novel therapeutics.Sorafenib inactivates STAT3 and STAT3-related signaling by inducing a conformational change in and releasing the autoinhibition of Src homology region 2 domaincontaining phosphatase-1.This phosphatase negatively regulates STAT3 activity,which leads to the subsequent apoptosis of cancer cells.The novel anti-cancer property of sorafenib will be discussed in this review,not only adding information regarding its mechanism of action but also providing an innovative approach for the development of cancer therapeutics in the future.

Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion

Signal transducer and activator of transcription（STAT）is a unique protein family that binds to DNA,coupled with tyrosine phosphorylation signaling pathways,acting as a transcriptional regulator to mediate a variety of biological effects.Cerebral ischemia and reperfusion can activate STATs signaling pathway,but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging（DWI）in rats after cerebral ischemia/reperfusion.Here,we established a rat model of focal cerebral ischemia injury using the modified Longa method.DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient.STAT3 protein expression showed no significant change after reperfusion,but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours.Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area.These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

Role of Toll-like receptor 4 and Janus kinase and signal transducer and activator of transcription signal transduction pathway in sepsis-induced brain damage

The Janus kinase and signal transducer and activator of transcription （JAK/STAT） signal transduction pathway is involved in sepsis-induced functional damage to the heart, liver, kidney, and other organs. However, the cellular and molecular mechanisms underlying sepsis-induced brain damage remain elusive. In the present study, we found severe loss of neurons in the hippocampal CA1 region in rats with sepsis-induced brain damage following intraperitoneal injection of endotoxin, The expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 was significantly increased in brain tissues following lipopolysaccharide exposure. AG490 （JAK2 antagonist） and rapamycin （STAT3 antagonist） significantly reduced neuronal loss and suppressed the increased expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 in the hippocampal CA1 region in sepsis-induced brain damaged rats. Overall, these data suggest that blockade of the JAK/STAT signal transduction pathway is neuroprotective in sepsis-induced brain damage via the inhibition of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 exoression.

Hepatitis C Virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-α through dephosphorylation

AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction

BACKGROUND Leukemia stem cells(LSCs)are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia(AML),as they are protected by the bone marrow microenvironment(BMM)against conventional therapies.Gossypol acetic acid(GAA),which is extracted from the seeds of cotton plants,exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2.AIM To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism.METHODS In this study,LSCs were magnetically sorted from AML cell lines and the CD34+CD38-population was obtained.The expression of leucine-rich pentatricopeptide repeat-containing protein(LRPPRC)and forkhead box M1(FOXM1)was evaluated in LSCs,and the effects of GAA on malignancies and mitochondrial RESULTS LRPPRC was found to be upregulated,and GAA inhibited cell proliferation by degrading LRPPRC.GAA induced LRPPRC degradation and inhibited the activation of interleukin 6(IL-6)/janus kinase(JAK)1/signal transducer and activator of transcription(STAT)3 signaling,enhancing chemosensitivity in LSCs against conventional chemotherapies,including L-Asparaginase,Dexamethasone,and cytarabine.GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC.Furthermore,GAA induced reactive oxygen species accumulation,disturbed mitochondrial homeostasis,and caused mitochondrial dysfunction.By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC,GAA resulted in the elimination of LSCs.Meanwhile,GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage.CONCLUSION Taken together,the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Glycine Attenuates Myocardial Fibrosis in Myocardial Infarction in Rats Partly through Modulating Signal Transducer and Activator of Transcription 3/Nuclear Factor-κB/Transforming Growth Factor-β axis

Objective: Inflammation and fibrosis are strongly associated with each other. Glycine is present in various traditional Chinese medicines and exhibits anti-inflammatory activity. However, the effects of glycine on myocardial fibrosis(MF) in rats with myocardial infarction(MI) have not been reported. The purpose of this study is to investigate the effects of glycine therapy on MF and comprehend its underlying mechanisms. Materials and Methods: Left anterior descending artery ligation-induced MI in Sprague Dawley rats was leveraged to assess the therapeutic effects of Glycine. Rats received either normal saline or glycine(0.5 mg/g bodyweight) for 7 days. Results: Glycine upregulated cardiac ejection fraction and fractional shortening to improve cardiac function, as evaluated by echocardiography. Histological and immunohistochemical analyses demonstrated that glycine could decrease inflammatory cell infiltration and alleviate collagen deposition. Western blotting revealed that nuclear factor-κB(NF-κB)-mediated inflammatory signaling was also downregulated by glycine treatment. The expression of signal transducer and activator of transcription 3(STAT3), tumor necrosis factor-α, and transforming growth factor-β(TGF-β) was decreased significantly in the glycine-treated group compared to the model group. Thus, glycine plays a protective role against myocardial ischemia and subsequent MF. Conclusion: The protective effects of glycine were achieved partly through STAT3/NF-κB/TGF-β signaling pathway.

Galectin 2 regulates JAK/STAT3 signaling activity to modulate oral squamous cell carcinoma proliferation and migration in vitro

Background:Galectin 2(LGALS2)is a protein previously reported to serve as a mediator of disease progression in a range of cancers.The function of LGALS2 in oral squamous cell carcinoma(OSCC),however,has yet to be explored,prompting the present study to address this literature gap.Methods:Overall,144 paired malignant tumor tissues and paracancerous OSCC patient samples were harvested and the LGALS2 expression levels were examined through qPCR and western immunoblotting.The LGALS2 coding sequence was introduced into the pcDNA3.0 vector,to enable the overexpression of this gene,while an LGALS2-specific shRNA and corresponding controls were also obtained.The functionality of LGALS2 as a regulator of the ability of OSCC cells to grow and undergo apoptotic death in vitro was assessed through EdU uptake and CCK-8 assays,and flow cytometer,whereas a Transwell system was used to assess migratory activity and invasivity.An agonist of the Janus Kinase 2(JAK2)/Signal Transducer and Activator of Transcription 3(STAT3)pathway was also used to assess the role of this pathway in the context of LGALS2 signaling.Results:Here,we found that lower LGALS2 protein and mRNA expression were evident in OSCC tumor tissue samples,and these expression levels were associated with clinicopathological characteristics and patient survival outcomes.Silencing LGALS2 enhanced proliferation in OSCC cells while rendering these cells better able to resist apoptosis.The opposite was instead observed after LGALS2 was overexpressed.Mechanistically,the ability of LGALS2 to suppress the progression of OSCC was related to its ability to activate the JAK/STAT3 signaling axis.Conclusion:Those results suggest a role for LGALS2 as a suppressor of OSCC progression through its ability to modulate JAK/STAT3 signaling,supporting the potential utility of LGALS2 as a target for efforts aimed at treating OSCC patients.

神经肌肉电刺激通过调控白细胞介素6/信号转导子及转录激活子3信号通路促进脊髓损伤后小鼠运动功能恢复

目的观察神经肌肉电刺激(NMES)对脊髓损伤后小鼠白细胞介素6(IL-6)/STAT3信号通路的影响,探讨其对运动功能恢复的机制。方法选取SPF级小鼠72只随机分成假手术(sham)组、脊髓损伤组(SCI)和NMES组。利用BMS评分、斜坡实验与电生理(EMG)评估小鼠脊髓损伤后肢体运动恢复情况。Western blotting和Real-time PCR检测各组小鼠脊髓组织中相关炎症因子、IL-6/STAT3信号通路与胶质纤维酸性蛋白(GFAP)和脑源性神经营养因子(BDNF)的表达。HE染色观察各组小鼠脊髓形态结构。结果NMES组BMS评分和小鼠斜坡实验均高于SCI组(P<0.05);NMES组小鼠运动诱发电位(MEP)最大振幅高于SCI组(P<0.05);NMES组脊髓组织TNF-α、IL-12A与GFAP表达量均低于SCI组(P<0.05),TGF-β、IL-10与BDNF表达量均高于SCI组(P<0.05);与SCI组相比,NMES组小鼠脊髓空洞较少,脊髓形态修复较好;与SCI组相比,NMES组IL-6/STAT3信号通路蛋白表达均低于SCI组(P<0.05)。结论神经肌肉电刺激通过抑制IL-6/STAT3信号通路发挥抗炎作用,从而促进脊髓损伤后小鼠后肢运动功能的恢复。

化瘀止痛贴膏调节IL-6/STAT3信号通路对急性软组织损伤大鼠炎症反应的影响

目的探讨化瘀止痛贴膏调节白细胞介素-6(IL-6)/信号转导与转录激活子3(STAT3)信号通路对急性软组织损伤(ASTI)大鼠炎症反应的影响。方法将SPF级SD大鼠随机分为对照组、模型组、化瘀止痛贴膏低、中、高剂量组、精制狗皮膏组。利用机械冲击挫伤法建立ASTI动物模型,建模成功后,各组贴敷相应药物,1次/d,每次给药8 h,连续7 d。对大鼠进行ASTI损伤评分;采用血液流变检测仪检测血液流变学指标;苏木精和伊红(HE)染色法观察大鼠损伤部位肌肉组织病理变化;酶联免疫吸附法(ELISA)检测各组大鼠损伤部位肌肉组织中IL-6、IL-β、肿瘤坏死因子-α(TNF-α)水平;实时荧光定量PCR(qRT-PCR)法检测损伤部位肌肉组织中IL-6与STAT3 mRNA表达;蛋白免疫印迹(Western blot)法检测大鼠损伤部位肌肉组织中IL-6/STAT3信号通路蛋白表达。结果与对照组比较,模型组大鼠软组织损伤评分、全血黏度、血浆黏度、红细胞压积、肌肉组织病理学评分、IL-6、IL-β、TNF-α水平、IL-6与STAT3 mRNA表达水平及IL-6、p-STAT3/STAT3蛋白表达水平升高(均P<0.05);与模型组比较,化瘀止痛贴膏低、中、高剂量组和狗皮膏药组大鼠软组织损伤评分、全血黏度、血浆黏度、红细胞压积、肌肉组织病理学评分、IL-6、IL-β、TNF-α水平、IL-6与STAT3 mRNA表达水平及IL-6、p-STAT3/STAT3蛋白表达水平降低(均P<0.05);化瘀止痛贴膏高剂量组与精制狗皮膏组大鼠以上指标差异均无统计学意义(P>0.05)。结论化瘀止痛贴膏可能通过下调IL-6/STAT3信号通路缓解ASTI大鼠的炎症反应。

匹莫齐特对脂多糖诱导RAW264.7细胞诱导型一氧化氮合成酶表达的调控及其作用机制

目的运用脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7细胞株,探究匹莫齐特(Pimozide)对一氧化氮和诱导型一氧化氮合成酶(iNOS)合成的影响和作用机制。方法用含10%胎牛血清、100 U·mL^(-1)的青链霉素DMEM培养液将RAW264.7细胞稀释为每孔2×105个接种于24孔板中进行培养,分为空白对照组(仅含DMEM培养液+RAW264.7细胞)、Pimozide组(仅含RAW264.7细胞和10μmol·L^(-1)Pimozide培养液)、LPS诱导(LPS 1 mg·L^(-1))组(LPS+RAW264.7细胞)、药物处理组[含细胞和不同浓度药物,包括Pimozide低(LPS+2.5μmol·L^(-1))、中(LPS+5μmol·L^(-1))、高(LPS+10μmol·L^(-1))组]。各组培养上清液中一氧化氮水平测定采用Griess法进行检测。采用实时定量聚合酶链反应(RT-PCR)法和免疫蛋白印迹法分别检测iNOS mRNA表达水平和iNOS和磷酸化的信号传导及转录激活因子通路-5的蛋白表达相对水平。结果用含10%胎牛血清、100 U·mL^(-1)的青链霉素DMEM培养液培养RAW264.7细胞24 h后,各组培养上清液一氧化氮表达水平差异有统计学意义(F=25.69,P<0.05);Pimozide低(LPS+2.5μmol·L^(-1))、中(LPS+5μmol·L^(-1))、高(LPS+10μmol·L^(-1))组一氧化氮释放的抑制率差异有统计学意义(F=132.49,P<0.05)。各组iNOS mRNA和蛋白水平表达差异有统计学意义(F=118.59和23.37,P<0.05),同时发现各组磷酸化的信号传导及转录激活因子通路-5/信号传导及转录激活因子通路-5比值差异有统计学意义(F=12.07,P<0.05)。结论Pimozide可抑制RAW264.7细胞中iNOS表达和一氧化氮的生成,其作用机制可能与抑制磷酸化的信号传导及转录激活因子通路-5的生成相关。

MK8719激活STAT6并促进抗炎型小胶质细胞活化在缺血性脑损伤大鼠中发挥保护作用的研究

目的:观察氧连接N-乙酰葡萄糖胺(O-linked N-acetylglucosamine,O-GlcNAc)水解酶抑制剂MK8719在脑缺血损伤大鼠中的作用。方法:建立大鼠大脑中动脉梗塞模型模拟脑缺血再灌注损伤,通过2,3,5-三苯基氯化四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC)染色评估脑梗死体积,并通过改良的神经损伤评分量表评估大鼠的神经运动障碍减轻,HE和尼式染色用于评估脑损伤后组织改变,组织免疫荧光染色用于评估抗炎型小胶质细胞活化情况。体外培养BV2小胶质细胞并建立氧糖剥夺/复糖复氧模型模拟缺血缺氧再灌注损伤,Western blotting检测总蛋白及核蛋白中的信号转导及转录激活蛋白6(signal transducer and activator of transcription 6,STAT6)、磷酸化STAT6及O-GlcNAc糖基化STAT6表达。酶联免疫吸附检测(Enzyme linked immunosorbent assay,ELISA)评估小胶质细胞释放的炎性因子白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-10(interleukin-10,IL-10)和转化生长因子-β(transforming growth factor-β,TGF-β)量。结果:MK8719给药组大鼠梗死灶体积减小,神经运动障碍,抗炎型小胶质细胞比例增加;在体外培养的小胶质细胞中,MK8719组的O-GlcNAc糖基化STAT6、磷酸化STAT6及核STAT6表达增加(P<0.001),促炎因子(IL-6及IL-1β)释放减少(P<0.001),抗炎因子(IL-10及TGF-β)释放增加(P<0.001)。结论:MK8719对脑缺血损伤大鼠有保护作用,其作用机制可能与STAT6激活诱导的抗炎型小胶质细胞活化有关。

Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion,migration,and angiogenesis in colorectal cancer

BACKGROUND As a novel endogenous anti-angiogenic molecule, vasohibin 1(VASH1) is not only expressed in tumor stroma, but also in tumor tissue. Moreover, studies have shown that VASH1 may be a prognostic marker in colorectal cancer(CRC). Knockdown of VASH1 enhanced transforming growth factor-β1(TGF-β1)/Smad3 pathway activity and type Ⅰ/Ⅲ collagen production. Our previous findings suggest that ELL-associated factor 2(EAF2) may play a tumor suppressor and protective role in the development and progression of CRC by regulating signal transducer and activator of transcription 3(STAT3)/TGF-β1 signaling pathway. However, the functional role and mechanism of VASH1-mediated TGF-β1 related pathway in CRC has not been elucidated.AIM To investigate the expression of VASH1 in CRC and its correlation with the expression of EAF2. Furthermore, we studied the functional role and mechanism of VASH1 involved in the regulation and protection of EAF2 in CRC cells in vitro.METHODS We collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein and VASH1 protein in patients with advanced CRC. Following, we investigated the effect and mechanism of EAF2 and VASH1 on the invasion, migration and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTS Our findings indicated that EAF2 was down-regulated and VASH1 was upregulated in advanced CRC tissue compared to normal colorectal tissue. KaplanMeier survival analysis showed that the higher EAF2 Level group and the lower VASH1 Level group had a higher survival rate. Overexpression of EAF2 might inhibit the activity of STAT3/TGF-β1 pathway by up-regulating the expression of VASH1, and then weaken the invasion, migration and angiogenesis of CRC cells.CONCLUSION This study suggests that EAF2 and VASH1 may serve as new diagnostic and prognostic markers for CRC, and provide a clinical basis for exploring new biomarkers for CRC. This study complements the mechanism of EAF2 in CRC cells, enriches the role and mechanism of CRC cellderived VASH1, and provides a new possible subtype of CRC as a therapeutic target of STAT3/TGF-β1 pathway.