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.

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.

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.

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.

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。

基于IL-6/JAK2/STAT3信号通路探讨灰树花提取物对溃疡性结肠炎大鼠结肠组织炎症反应的影响

目的:探讨灰树花提取物通过调控白细胞介素6(IL-6)/蛋白酪氨酸激酶2(JAK2)/信号转导和激活因子3(STAT3)信号通路,对溃疡性结肠炎(UC)大鼠结肠组织炎症反应的影响及作用机制。方法:将40只SD大鼠随机分为空白对照组、UC模型组、灰树花治疗组、西药治疗组、联合治疗组,每组8只。采用3%DSS自由饮用法7 d建立UC大鼠模型后,治疗组分别灌胃灰树花提取物10 mg/(kg·d)、柳氮磺吡啶0.3 g/(kg·d)及等量两种药物,连续14 d。实验期间观察大鼠一般状态,计算疾病活动指数(DAI)评分;采用HE染色法观察大鼠结肠组织病理改变;Western blot检测大鼠结肠组织IL-6、JAK2、STAT3、pSTAT3蛋白表达水平;ELISA检测大鼠血清中IL-6含量;免疫组化法测定大鼠结肠组织中IL-6R、MPO蛋白含量及表达情况。结果:与空白对照组比较,UC模型组大鼠一般状态欠佳,DAI评分升高,HE染色可见明显的组织黏膜损伤与炎症细胞浸润;结肠组织IL-6、JAK2、STAT3、p-STAT3蛋白表达水平及IL-6R、MPO含量均显著升高(P<0.01);血清中IL-6含量显著升高(P<0.01),差异有统计学意义。与UC模型组比较,各治疗组大鼠的一般状态均有所改善,DAI评分降低,HE染色可见黏膜损伤有不同程度改善,偶见炎症细胞浸润;结肠组织IL-6、JAK2、STAT3、p-STAT3蛋白表达水平显著降低(P<0.01),结肠组织IL-6R和MPO含量及血清中IL-6含量均明显减少(P<0.01或P<0.05),差异有统计学意义。结论:灰树花提取物可通过调控IL-6/JAK2/STAT3信号通路相关因子表达,减轻UC大鼠结肠组织炎症反应。

Three important components in the regeneration of the cavernous nerve： brain-derived neurotrophic factor, vascular endothelial growth factor and the JAK/STAT signaling pathway

Retroperitoneal operations, such as radical prostatectomy, often damage the cavernous nerve, resulting in a high incidence of erectile dysfunction. Although improved nerve-sparing techniques have reduced the incidence of nerve injury, and the administration of phosphodiesterase type 5 inhibitors has revolutionized the treatment of erectile dysfunction, this problem remains a considerable challenge. In recent years, scientists have focused on brain-derived neurotrophic factor and vascular endothelial growth factor in the treatment of cavernous nerve injury in rat models. Results showed that both compounds were capable of enhancing the regeneration of the cavernous nerve and that activation of the Janus kinase （JAK）/signal transducer and activator of transcription （STAT） pathway played a major role in the process.

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。

基于IL-6/JAK2/STAT3信号轴研究阳和平喘颗粒调控哮喘大鼠气道重塑作用机制

目的:研究阳和平喘颗粒对哮喘大鼠气道重塑及白细胞介素-6(IL-6)/Janus蛋白酪氨酸激酶2(JAK2)/信号转导和转录活化因子3(STAT3)信号轴在其中的作用机制。方法:选取健康雄性SD大鼠42只,随机数字法分为正常对照组7只与造模组35只,造模组采用卵清蛋白(OVA)联合氢氧化铝腹腔注射2周的方式进行致敏,正常对照组采用等量的生理盐水;2周后将造模组随机分为模型组、阳和平喘高、中、低剂量组和地塞米松组,每组7只;后4周采用OVA雾化+灌胃的方式进行激发和治疗,阳和平喘高中低组每日分别予以15.48、7.74、3.87 g/kg阳和平喘颗粒灌胃,地塞米松组予以0.0625 mg/kg地塞米松进行灌胃,其余组灌胃等量生理盐水。HE、PAS、Masson染色观察大鼠肺组织病理学变化;ELISA检测大鼠血清中IL-6、IL-23、IL-17A水平;Western blot检测肺组织中JAK-2、P-JAK2、STAT3、P-STAT3蛋白表达量;qRT-PCR检测大鼠肺组织中IL-6、JAK2、STAT3的mRNA水平。结果:与正常对照组比较,模型组大鼠肺组织有大量炎性细胞浸润,杯状细胞增生、上皮下胶原纤维沉积、气道上皮增厚较为明显;血清中IL-6、IL-23、IL-17A水平显著升高(P<0.01),肺组织JAK-2、P-JAK2、STAT3、P-STAT3的蛋白表达量和IL-6、JAK2、STAT3的mRNA表达水平显著升高(P<0.01);与模型组比较,各给药组炎性细胞浸润、杯状细胞增生、上皮下胶原纤维沉积、气道上皮增厚程度明显减轻,血清中IL-6、IL-23、IL-17A水平显著降低(P<0.01),肺组织JAK-2、P-JAK2、STAT3、P-STAT3的蛋白表达量和IL-6、JAK2、STAT3的mRNA水平显著降低(P<0.01)。结论:阳和平喘颗粒可明显缓解哮喘大鼠气道重塑,其机制可能是通过抑制IL-6/JAK2/STAT3信号轴发挥作用。

Differential regulation of JAK/STAT-signaling in patients with ulcerative colitis and Crohn’s disease

In 2018,the pan-Janus kinase(JAK)inhibitor tofacitinib was launched for the treatment of ulcerative colitis(UC).Although tofacitinib has proven efficacious in patients with active UC,it failed in patients with Crohn’s disease(CD).This finding strongly hints at a different contribution of JAK signaling in both entities.Here,we review the current knowledge on the interplay between the JAK/signal transducer and activator of transcription(STAT)pathway and inflammatory bowel diseases(IBD).In particular,we provide a detailed overview of the differences and similarities of JAK/STAT-signaling in UC and CD,highlight the impact of the JAK/STAT pathway in experimental colitis models and summarize the published evidence on JAK/STAT-signaling in immune cells of IBD as well as the genetic association between the JAK/STAT pathway and IBD.Finally,we describe novel treatment strategies targeting JAK/STAT inhibition in UC and CD and comment on the limitations and challenges of the new drug class.