Oncolytic Newcastle disease virus inhibits IL-6 induced migration and invasion of human glioblastoma U87MG cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨溶瘤新城疫病毒（NDV）对IL-6诱导的人胶质母细胞瘤U87MG细胞增殖、迁移和侵袭的作用及其可能的机制。方法：将U87MG细胞分为对照组、IL-6组、NDV组、NDV+IL-6组，其中IL-6组与NDV+IL-6组用75 ng/mL IL-6预处理1 h，其余组用DMEM预处理1 h，后分别用DMEM、75 ng/mL IL-6、1 HU NDV、1 HU NDV+75 ng/mL IL-6处理24 h。MTT法、细胞划痕实验和Transwell侵袭实验分别检测IL-6、NDV对U87MG细胞增殖、迁移和侵袭的影响，WB法检测各组细胞JAK2、p-JAK2、STAT3、p-STAT3和MMP2蛋白的表达水平。结果：与对照组相比，IL-6组细胞迁移率显著升高（P<0.05），侵袭细胞数目显著增多（P<0.01）；与IL-6组相比，NDV+IL-6组U87MG细胞增殖率显著降低（P<0.05），细胞迁移率和侵袭细胞数目均显著降低（均P<0.01）。WB实验结果显示，与对照组相比，IL-6组p-STAT3/STAT3比值显著升高（P<0.01），NDV组p-JAK2/JAK2、p-STAT3/STAT3比值显著降低（P<0.05，P<0.01），MMP-2蛋白表达量显著降低（P<0.01）；与IL-6组相比，NDV+IL-6组p-STAT3/STAT3比值、MMP-2蛋白表达量均显著降低（均P<0.05）。结论：NDV能抑制IL-6对人脑胶质瘤U87MG细胞迁移和侵袭的诱导作用，其机制可能与JAK2/STAT3信号通路的参与调控有关。

Effects of α-hederin alone or in combination with cisplatin on the proliferation and apoptosis of non-small cell lung cancer cells based on EGFR/AKT and JAK2/STAT3 pathways / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨α-常春藤皂苷（α-Hed）诱导非小细胞肺癌（NSCLC）细胞凋亡的作用靶点及其潜在机制，明确α-Hed与顺铂（DDP）联用后对相应的靶点蛋白表达的影响。方法：采用CCK-8法检测不同浓度α-Hed处理后NSCLC细胞A549、H1299和PC-9的存活率，采用Annexin Ⅴ-FITC/PI染色流式细胞术检测细胞凋亡率，采用WB法检测细胞中C-caspase-3和Bcl-2蛋白的表达。通过网络药理学相关方法筛选α-Hed的潜在靶点，利用分子对接法分析其结合效果，WB法检测靶点蛋白的表达。通过CCK-8法、细胞集落形成实验和WB法检测α-Hed与DDP联用对NSCLC细胞的抑制作用。结果：给药24和48 h后，10、15和20 μmol/L α-Hed可以显著抑制NSCLC细胞增殖活力（均P<0.01）；与对照组相比，20 μmol/L α-Hed处理后细胞凋亡率显著升高（P<0.01）；α-Hed可上调NSCLC细胞中C-caspase-3的表达（P<0.05），下调Bcl-2的表达（P<0.05）。网络药理学和分子对接筛选出结合亲和力小于-5 kcal/mol的靶点AKT1、STAT3、EGFR和JAK2。WB法检测结果显示，α-Hed处理后A549、H1299细胞中EGFR、p-AKT/AKT、p-STAT3/STAT3和JAK2蛋白的表达均明显下调（均P<0.05）。α-Hed与DDP联用后，更显著地抑制NSCLC细胞的增殖（P<0.01），进一步下调EGFR、p-AKT/AKT、p-STAT3/STAT3和JAK2蛋白的表达（P<0.05或P<0.01）。结论：α-Hed通过下调EGFR和JAK2的表达抑制STAT3和AKT的磷酸化，诱导NSCLC细胞凋亡，与DDP联用后其抑制效果增强，EGFR/AKT和JAK2/STAT3通路也进一步被抑制。

Mechanism of Dihuangyin in Treatment of Mice with Atopic Dermatitis by Regulating JAK1/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and potential mechanism of Dihuangyin on 2， 4-dinitrochlorobenzene （DNCB） -induced model mice with atopic dermatitis （AD）. MethodA mouse model with AD was established by repeatedly stimulating the back skin of mice with DNCB. After successful modeling， the mice were randomly divided into model group， Runzao group （0.78 g·kg-1）， and high， medium， and low dose （40.30， 20.15， and 10.08 g·kg-1） groups of Dihuangyin， with 12 mice in each group， and the blank group consisted of 12 mice， 72 in total. The administration groups were given the corresponding liquid by dose， and the blank group and model group were given the same dose of pure water by intragastric administration， once a day. The skin lesions and scratching times of mice were observed after continuous administration for two weeks. The back skin lesions of mice were stained with hematoxylin-eosin （HE） and toluidine blue to observe the pathology. The contents of serum immunoglobulin E （IgE）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， and interferon-γ （IFN-γ） were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression levels of IFN-γ， IL-4， IL-6， Janus kinase 1 （JAK1）， and transcriptional activator 3 （STAT3） in skin lesion tissue were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expressions of JAK1， phosphorylation（p）-JAK1， STAT3， and p-STAT3 proteins in skin lesion tissue were detected by Western blot. ResultCompared with the blank group， the back skin of the model group showed large-scale scab， dryness， erosion， hypertrophy with scratching， epidermal hyperplasia with hyperkeratosis and parakeratosis， hyperacanthosis with edema， and a large number of mast cell infiltration in the dermis， some of which were degranulated. The contents of IgE， IL-4， IL-6， and IFN-γ in the serum of mice were significantly increased （P<0.01）， and the protein expression levels of p-JAK1， STAT3， and p-STAT3 and mRNA expressions of IL-4， IL-6， IFN-γ， JAK1， and STAT3 in skin lesion tissue were significantly increased （P<0.01）. Compared with the model group， only a small amount of dryness and desquamation were observed in the back skin of mice in each administration group， and cell edema was reduced. The inflammatory infiltration was significantly reduced， and the number of mast cell infiltration was significantly decreased. The serum IgE， IL-4， IL-6， and IFN-γ of mice were decreased to varying degrees （P<0.05， P<0.01）. The protein expression levels of p-JAK1， STAT3， and p-STAT3 and mRNA expressions of IL-4， IL-6， IFN-γ， JAK1， and STAT3 in skin lesion tissue were significantly decreased， and the effect of high dose group of Dihuangyin was the best （P<0.01）. ConclusionDihuangyin can improve skin lesions and pruritus in mice with AD， and its mechanism may be related to the effective regulation of cytokines on the helper T cells （Th1）/Th2 axis by interfering with the JAK1/STAT3 signaling pathway and affecting skin barrier function.

Effect of Qihuang Jianpi Zishen Granules on STAT3/mTOR Signaling Pathway in Patients with Systemic Lupus Erythematosus / 中国实验方剂学杂志

ObjectiveTo investigate the clinical efficacy of Qihuang Jianpi Zishen Granules in the treatment of systemic lupus erythematosus （SLE） and its effect on the signal transducer and activator of tranSCription 3/mammalian target of rapamycin （STAT3/mTOR） signaling pathway， and to decipher the possible mechanism. MethodSixty female SLE patients who met the criteria in the First Affiliated Hospital of Anhui University of Chinese Medicine from May 2022 to May 2023 were selected and randomized into a control group and an observation group （30 cases in each group）. The control group was treated with prednisone acetate + hydroxychloroquine sulfate orally， and the observation group was additionally treated with Qihuang Jianpi Zishen granules. The treatment lasted for 8 weeks. The SLE disease activity （SLEDAI）， TCM syndrome score， erythrocyte sedimentation rate （ESR）， hypersensitive C-reactive protein （hs-CRP）， immune indexes ［immunoglobulin G （IgG）， C3， C4， CD4+， and CD8+］， interleukin （IL）-17， IL-23， interferon （IFN）-γ， 24 h urinary protein （24 h PRO）， serum creatinine （SCr）， and expression of proteins ［STAT3， phosphorylated （p）-STAT3， mTOR protein and STAT3，mTOR mRNA］ in the STAT3/mTOR signaling pathway were determined before and after treatment. In addition， the adverse reactions were recorded. ResultAfter 8 weeks of treatment， the total response rate in the observation group was 93.33% （28/30）， which was higher than that （70.00%， 21/30） in the control group （χ2=4.007， P<0.05）. After treatment， both groups showed declined SLEDAI， TCM syndrome score， ESR， hs-CRP， IgG， CD8+， IL-17， IL-23， IFN-γ， 24 h PRO， SCr， and expression of proteins in the STAT3/mTOR pathway （P<0.01） and elevated levels of C3， C4， and CD4+ （P<0.01）. Moreover， the observation group had lower SLEDAI， TCM syndrome score， ESR， hs-CRP， IgG， CD8+， IL-17， IL-23， IFN-γ， 24 h PRO， SCr， and expression of proteins in the STAT3/mTOR pathway （P<0.05， P<0.01） and higher levels of C3， C4， and CD4+ （P<0.05， P<0.01） than the control group after treatment. Neither group showed serious adverse reactions during the treatment period. ConclusionQihuang Jianpi Zishen Granules can ameliorate the inflammatory response， reduce the disease activity， and mitigate the kidney injury in SLE by inhibiting the STAT3/mTOR signaling pathway to regulate the immune function.

Targeting FAPα-positive lymph node metastatic tumor cells suppresses colorectal cancer metastasis

Lymphatic metastasis is the main metastatic route for colorectal cancer, which increases the risk of cancer recurrence and distant metastasis. The properties of the lymph node metastatic colorectal cancer (LNM-CRC) cells are poorly understood, and effective therapies are still lacking. Here, we found that hypoxia-induced fibroblast activation protein alpha (FAPα) expression in LNM-CRC cells. Gain- or loss-function experiments demonstrated that FAPα enhanced tumor cell migration, invasion, epithelial-mesenchymal transition, stemness, and lymphangiogenesis via activation of the STAT3 pathway. In addition, FAPα in tumor cells induced extracellular matrix remodeling and established an immunosuppressive environment via recruiting regulatory T cells, to promote colorectal cancer lymph node metastasis (CRCLNM). Z-GP-DAVLBH, a FAPα-activated prodrug, inhibited CRCLNM by targeting FAPα-positive LNM-CRC cells. Our study highlights the role of FAPα in tumor cells in CRCLNM and provides a potential therapeutic target and promising strategy for CRCLNM.

Hsa_circ_0013561 promotes progression of nasopharyngeal carcinoma by activating JAK2/STAT3 signaling pathway

Abstract Objective Nasopharyngeal Carcinoma (NPC) is a malignancy of epithelium of epithelium of the nasopharynx, with the highest incidence of otolaryngeal malignancies. A growing number of studies confirm that Circular RNA (circRNA) plays an important role in tumor development, including Hsa_circ_0013561. This study aims to elucidate the process and mechanism of NPC regulation hsa_circ_0013561. Methods In this study, circRNA expression nodes and subcellular localization in NPC tissues were analyzed by fluorescence in situ hybridization. The expression of hsa_circ_0013561 in NPC cells was further clarified by RT-qPCR. At the same time, the lentivirus vector interfered by hsa_circ_0013561 was constructed and transfected. The cell proliferation was detected by CCK-8 method, EdU assay and plate cloning assay. The cell cycle and apoptosis were detected by flow cytometry, and the cell migration ability was detected by wound healing assay and Transwell assay. Western blotting examined the expression of apoptosis, Epithelial-Mesenchymal Transition (EMT)-associated proteins, and Janus Kinase/Signal Transductor and Activator of Transcription (JAK/STAT) signaling pathway-related proteins. Results The results showed that the expression of hsa_circ_0013561 in NPC samples was significantly upregulated and hsa_circ_0013561 localized in the cytoplasm. After down-regulating hsa_circ_0013561 expression, it significantly inhibited the proliferation and metastasis ability of NPC, inhibited EMT progression, and promoted apoptosis. Further studies showed that interference hsa_circ_0013561 significantly inhibited JAK2/STAT3 signaling pathway activation and induced the expression of apoptosis-related proteins. Conclusion In summary, we found that hsa_circ_0013561 is a pro-tumor circRNA in NPC, which can reduce the activation of JAK2/STAT3 pathway by knocking down hsa_circ_0013561, thereby slowing down the malignant progression of NPC. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 4.

Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway / 中国中药杂志

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.

Inhibitory effect of miR-497 on corneal neovascularization and its regulation mechanism targeting STAT3 / 中华实验眼科杂志

Objective:To investigate the role of microRNA (miR)-497 in the formation of corneal neovascularization (CNV) induced by alkali burn and its mechanism.Methods:Forty-two wild type (WT) C57BL/6 mice aged 6 to 8 weeks, 42 CRISPR/Cas9 mediated miR-497 knockout (KO) and 42 CRISPR/Cas9 mediated overexpression transgenic (TG) C57BL/6 mice were selected and assigned as WT group, KO group and TG group, respectively.The corneal alkali burn model was established.At 3, 7, 14 and 21 days after modeling, corneal epithelium damage and stromal turbidity were scored according to slit lamp microscopy.The area of neovascularization was measured.Corneal structural changes and expression of inflammatory cells were observed by histopathological staining.The expression of CD31 in corneal tissues was detected by immunohistochemistry staining.The targeted binding relationship between miR-497 and signal transducer and activator of transcription 3 (STAT3) was detected by luciferase reporter assay.The relative expressions of miR-497, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and macrophage inflammatory protein (MCP)-1 mRNA were detected by real-time quantitative PCR.At 14 days following modeling, the expression of STAT3 and p-STAT3 proteins in mice corneal tissues was detected by Western blot.The use and care of animals complied with the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No.2019K-K010).Results:Corneal injury, inflammatory cell infiltration and CNV occurred in mice cornea after alkali burn.Corneal epithelial injury score, corneal stromal turbidity score and CNV area increased first and reached the peak on the 14th day after modeling, and then decreased.There were significant differences in corneal epithelial injury score, corneal stromal turbidity score, CNV area and number of CD31-positive cells among various time points after alkali burn ( Fgroup=49.19, 34.56, 44.56, 77.56; all at P<0.01; Ftime=51.62, 65.62, 71.32, 46.12; all at P<0.01). Corneal epithelial injury score, corneal stromal turbidity score, CNV area and the number of CD31-positive cells were greater in KO group at various time points than in WT and TG groups, and those in WT group were greater than in TG group (all at P<0.05). In WT STAT3 co-transfected cells, the luciferase activity of the miR-497 group was significantly lower than that of the miR-negative control group and normal control group (both at P<0.05). In mutant STAT3-transfected cells, there was no significant difference in luciferase activity among all groups ( F=0.69, P=0.56). On the 14th day after modeling, the relative expression levels of miR-497 in corneal tissue of WT, KO and TG groups were 0.68±0.11, 0.41±0.06 and 1.05±0.14, respectively, which were significantly lower than 1.00±0.04, 0.56±0.07 and 1.34±0.11 before modeling (all at P<0.01). The relative expressions of STAT3 and p-STAT3 were higher in KO group than in WT and TG groups, and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.05). The expressions of VEGFA, TNF-α, IL-6, IL-1β and MCP-1 mRNA at various time points after modeling in various groups were significantly higher than before modeling, which were higher in KO group than in WT and TG groups and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.01). Conclusions:MiR-497 inhibits corneal inflammation and CNV formation induced by alkali burn.It might inhibit the activation of the inflammation signal pathway via targeting STAT3.

An update on signal transduction and activator of transcription 3-hyper-IgE syndrome / 中华实用儿科临床杂志

Hyper-IgE syndrome (HIES) comprises a group of rare primary immunodeficiencies, which are characterized by extremely high serum IgE levels, eczema, recurrent skin and pulmonary infections.Signal transduction and activator of transcription 3( STAT3)-HIES is the most common type, which is caused by dominant-negative mutations in STAT3.STAT3-HIES confers broad innate and acquired immune defects, defects in skeletal, connective tissue, and vascular functions, causing a clinical phenotype including eczema, staphylococcal and fungal skin and pulmonary infections, scoliosis and minimal trauma fractures, vascular tortuosity and aneurysm.In this article, the advance in diverse clinical manifestations and management strategies of STAT3-HIES was summarized.

Effects of mild hypothermia on microglia polarization and JAK2/STAT3 signaling pathway during cerebral ischemia-reperfusion in rats / 中华麻醉学杂志

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.

Scutellaria Baicalensis Polysaccharides Alleviates the Inflammation of DSS-induced UC Model Mice by Regulating JAK2/STAT3 Pathway and IL-23/IL-17 Inflammatory Axis / 中山大学学报(医学科学版)

ObjectiveTo investigate the effect and mechanism of Scutellaria Baicalensis polysaccharides on intestinal immunity in mice with ulcerative colitis （UC）. MethodsC57BL/6 mice were randomly assigned to blank group， model group， mesalazine group and Scutellaria Baicalensis polysaccharides low-dose， medium-dose and high-dose groups （n=10 mice/group）. The daily status of mice was observed and the disease activity index （DAI） score was recorded. The expression of cytokines including IL-6， IL-17 and IL-23 in serum was detected by ELISA. After the mice were sacrificed， HE was used to observe the intestinal mucosal damage in mice， and the colonic tissue damage index （TDI） score was recorded. Western blot and immunohistochemistry were used to detect the expression levels of Janus kinase 2 （JAK2）， signal transducer and activator of transcription （STAT3）， p-JAK2 and p-STAT3 proteins. ResultsCompared with the blank group， the DAI score of the model group was significantly increased， the contents of IL-6， IL-17 and IL-23 in serum were significantly increased， the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in colon tissue was significantly increased， and the expression levels of p-JAK2 and p-STAT3 were significantly increased. Compared with the model group， the DAI score of mice in each administration group was significantly decreased. The contents of IL-6， IL-17 and IL-23 in serum were decreased， the TDI score was decreased， the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in colon tissue was decreased， and the expression of p-JAK2 and p-STAT3 was decreased. ConclusionScutellaria Baicalensis polysaccharides may improve the inflammatory effect of UC mice through JAK2/STAT3 pathway and IL-23/IL-17 inflammatory axis.

Inhibitory effects of ursolic acid on IL-6-mediated invasion and migration of breast cancer cells / 中国药房

OBJECTIVE To investigate the inhibitory effects of ursolic acid on interleukin-6 （IL-6）-mediated invasion and migration of breast cancer MDA-MB-231 cells （hereinafter referred to as “231 cells”）. METHODS The effects of 20， 40， 80， 160 and 320 µmol/L ursolic acid on the proliferation rate of 231 cells were measured by CCK-8 method. The breast cancer 231 cells were divided into control group， model group and administration group. The migration and invasion abilities of cells were detected by scratch assay and Transwell assay. Real-time quantitative polymerase chain reaction （q-PCR） assay and Western blot assay were used to detect the mRNA and protein expressions of epithelial-mesenchymal transition-related makers such as E cadherin （E-cad）， matrix metalloprotein 2 （MMP2）， MMP9， vimentin （Vim）， CD44 molecule （CD44） and aldehyde dehydrogenase 1 family member A1 （ALDH1A1）. The phosphorylation levels of JAK2 and STAT3 in the Janus kinase 2/signal transducer and activator of transcription 3 （JAK2/STAT3） signaling pathway （in terms of p-JAK2/JAK2 ratio and p-STAT3/STAT3 ratio） were detected by Western blot assay. RESULTS A low concentration of ursolic acid of 20 µmol/L （no significant inhibitory effect on cell proliferation ability） was selected as the subsequent administration concentration. Compared with the control group， the migration and invasion abilities of cells in the model group were significantly enhanced （P＜0.05）； compared with the model group， the migration and invasion abilities of cells in the administered group were significantly reduced （P＜0.05）. Compared with the control group， the relative mRNA and protein expressions of epithelial-mesenchymal transition-related markers MMP9， MMP2， Vim， ALDH1A1 and CD44 were all elevated to different extents， and the mRNA and protein expressions of E-cad were all decreased to different extents in the model group cells， and part of the differences had statistical significance （P＜0.05）， the p-JAK2/JAK2 ratio and p-STAT3/STAT3 ratio were significantly increased in the model group （P＜0.05）； compared with the model group， the expressions of the above indicators were reversed to some extent in the administration group. CONCLUSIONS Ursolic acid blocks the activation of JAK2/STAT3 signaling pathwby the inflammatory factor IL-6， which ultimately interrupts the invasion and metastasis of breast cancer cells.

Influence of matrine on Th17/Treg balance in rats with inflammatory bowel disease by regulating IL-6/STAT3/NF-κB signaling pathway / 西安交通大学学报(医学版)

【Objective】 To investigate the influence of matrine (MT) on the balance of T helper cell 17 (Th17)/regulatory T cells (Treg) in rats with inflammatory bowel disease by regulating interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3)/nuclear transcription factor-κB (NF-κB) pathway. 【Methods】 SD rats were grouped into control check group (CK group), model group, low-dose MT group (MT-L group, 50 mg/kg), medium-dose MT group (MT-M group, 100 mg/kg), high-dose MT group (MT-H group, 200 mg/kg), mesalazine group (MSLM group, 0.42 g/kg), and MT-H+rIL-6 (IL-6 activator) group (200 mg/kg+0.05 mg/kg) according to the random number table method, with 18 in each group. Except for the CK group, the rats in other groups all received with 5% trinitrobenzenesulfonic acid (20 mg/kg) buffer solution mixed with 50% ethanol at a ratio of 1∶1 and then enema to construct a rat model of inflammatory bowel disease. After the successful modeling, they were treated with drug administration once a day for 7 weeks. The body weight of rats was measured at 1, 3, 5, and 7 weeks of administration; the changes of colon length of rats in each group were compared; HE staining was used to detect the pathological damage of rat colon tissue; the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-17 and IL-10 in serum of rats were detected by ELISA; the proportions of Th17 and Treg cells in peripheral blood of rats were detected by flow cytometry; Western blottingt was performed to detect the protein expression of retinoic acid-related orphan receptor γt (RORγt), forkhead box protein P3 (Foxp3), IL-6, p-STAT3, and p-NF-κB p65 in rat colon tissue. 【Results】 Compared with the CK group, the colon tissue of the model group was severely damaged by pathology, and the body weight (at 3, 5, and 7 weeks), the level of IL-10, the proportion of Treg cell, and the expression of Foxp3 protein were decreased, the colon length shortened, the levels of TNF-α, IL-17, the proportions of Th17 cell, Th17/Treg ratio, and the protein expression of RORγt, IL-6, p-STAT3, and p-NF-κB p65 increased (P<0.05). Compared with the model group, the corresponding indicators of the MT-L group, MT-M group, MT-H group, and MSLM group had the opposite trends (P<0.05); rIL-6 attenuated the promoting effect of high-dose MT on Th17/Treg balance in inflammatory bowel disease rats. 【Conclusion】 MT may promote Th17/Treg balance in inflammatory bowel disease rats by inhibiting IL-6/STAT3/NF-κB signaling pathway.

AG490 regulates JAK2/STAT3 pathway in M2-like macrophages via promoting the proliferation of gastric cancer cells and inhibiting apoptosis / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of JAK2/STAT3 signaling pathway inhibitor AG490 on the functional phenotype of M2-like macrophages and its effects on proliferation, apoptosis, migration, and invasion of gastric cancer cells. 【Methods】 Human mononuclear cell line THP-1 was induced to differentiate into M0 macrophages by PMA in vitro. The M1-like phenotype was induced by LPS and IFN-γ, and M2-like phenotype was induced by IL-4 and IL-13, respectively, and identified by immunofluorescence labeling CD68, CD86 and CD206. The mRNA expressions of CD163, Arg1, CCL22, PPARγ, IL-10, IL-20 and TNF-α were determined by RT-qPCR. The expressions of key proteins in JAK2/STAT3 signaling pathway were detected by Western blotting. M2-like macrophages were treated with JAK2/STAT3 inhibitor (AG490) to observe the expression level of marker genes for M2 like phenotype. Macrophages were co-cultured with gastric cancer cells, and the effects of the macrophages on proliferation, migration, invasion, and apoptosis of gastric cancer cells were detected by CCK-8 method, healing assay, transwell intracellular Matrigel invasion assay, and flow cytometry. The xenograft tumor model of MKN45 gastric cancer in nude mice was prepared, and the tumor size and quality were observed for 20 days after the model was established. 【Results】 THP-1 cells were induced into M1-like macrophages and M2-like macrophages. M1-like marker (CD86) and M2-like marker (CD206) were identified by flow cytometry. The P38MAPK, JAK2, p-STAT3/STAT3 protein levels of M2-like macrophages treated with AG490 were significantly reduced. The mRNA expression levels of Arg1, CCL22, PPARγ and IL-10 were significantly reduced in the group of M2-like macrophages treated with AG490. Co-culture of M2-like macrophages with gastric cancer cells could promote gastric cancer cell viability, increase migration and invasion ability, and inhibit apoptosis. When the group of M2-like macrophages treated with AG490 was co-cultured with gastric cancer cells, the proliferation activity of MKN-45 cells and MGC823 was significantly lower than that in M2 group (1.047±0.062 vs. 1.426±0.076, 1.149±0.006 vs. 1.301±0.015). Compared to M2 group, the migration (100.0%±5.73% vs. 72%±3.85%) and invasion ability (100.0%±7.40% vs. 60%±6.54%) of MGC823 gastric cancer cells in AG490 treatment group were significantly reduced. The apoptosis rate of MGC823 cells in the AG490 treated group was significantly higher than that in M2 group (27.51%±0.70% vs. 20.82%±0.92%). In the nude mouse xenograft tumor model, the volume and weight of the transplanted tumor collected at day 20 were significantly lower in AG490 treated group than in M2 group (736.04±182.34 vs. 1 080.5±250.57)mm3, (0.64±0.11 vs. 0.87±0.17)g. 【Conclusion】 AG490 downregulates the activation level of JAK2/STAT3 signaling pathway in M2-like macrophages, inhibits M2-type polarization, partially reverses the cancer-promoting function of M2-like macrophages, inhibits proliferation, migration and invasion of gastric cancer cells, and induces apoptosis. The JAK2/STAT3 signaling pathway can be further studied as a potential therapeutic target for gastric cancer.

Role of LIF/LIFR/STAT3 pathway in endometrial receptivity in rats with polycystic ovary syndrome / 西安交通大学学报(医学版)

【Objective】 To investigate the role of LIF/LIFR/STAT3 pathway in endometrial receptivity in rats with polycystic ovary syndrome (PCOS). 【Methods】 Forty 21-day-old SD female rats were divided into normal (control) group, model group, sham-operation group, and LIF group with 10 rats in each. The rat model of PCOS was constructed by subcutaneous injection of prasterone sodium sulfate at the back of the neck. The serum levels of testosterone (T), glucose and insulin in each group were detected. The morphological changes of the uterus in each group were observed by HE staining, and the morphological changes of endometrium were measured. Immunohistochemistry, Western blotting, and Real-time fluorescence quantitative PCR(qRT-PCR) were used to determine the protein expression and mRNA expression of LIF and STAT3 in rat endometrium. 【Results】 Compared with control group, the levels of integrin avb3, serum T, insulin and glucose in PCOS rats were significantly increased (P=0.000, P=0.000, P=0.001). Supplementation of exogenous LIF could significantly reduce the levels of integrin avb3, serum T, glucose and insulin in PCOS rats (P=0.000, P=0.002, P=0.003, P=0.007). HE results showed that exogenous LIF could reduce uterine cavity and glandular morphology in PCOS rats and increase the equivalent diameter (P=0.000, P=0.000) and area (P=0.000, P=0.000) of uterine glands and glandular cavity, the ratio of glandular interstitial area (P=0.000), and the average endometrial thickness (P=0.006), with statistically significant differences. Immunohistochemistry, Western blotting, and qRT-PCR results showed that the expression levels of LIF and p-STAT3 protein and mRNA in model group were significantly decreased compared with control group. Compared with model group, the protein and mRNA expressions of LIF and p-STAT3 in LIF group were significantly increased (P<0.05). 【Conclusion】 Exogenous LIF supplementation can improve endometrial receptivity in PCOS rats, and its mechanism is related to the LIF/LIFR/STAT3 pathway.

Influence of Limonin on Malignant Biological Behavior of Non-small Cell Lung Cancer Cells by Regulating JAK2/STAT3 Signaling Pathway / 肿瘤防治研究

Objective To investigate the influence of limonin on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells by regulating the protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods CCK-8 method was applied to detect the survival rate of A549 cells treated with different concentrations of limonin (0, 5, 10, 25, 50, 75, 100 μmol/L). A549 cells were separated into normal culture (NC) group, low-dose limonin group (treatment with 10 μmol/L limonin for 24 h), medium-dose limonin group (treatment with 25 μmol/L limonin for 24 h), high-dose limonin group (treatment with 50 μmol/L limonin for 24 h), coumermycin A1 group (treatment with 10 μmol/L JAK2 activator coumermycin A1+50 μmol/L limonin for 24 h), and AG490 group (treatment with 10 μmol/L JAK2 inhibitor AG490+50 μmol/L limonin for 24 h). Clone formation assay was applied to detect the clones of each group of cells. Transwell assay was applied to detect cell migration and invasion, and flow cytometry was applied to detect apoptosis. Western blot analysis was applied to detect the protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, E-cadherin, N-cadherin, and vimentin in each group. Results The viability of A549 cells decreased significantly in a limonin concentration-dependent manner (P < 0.05), with IC50 of 45.16±1.66 μmol/L. Concentrations of 10, 25, and 50 μmol/L were selected for subsequent experiments. The numbers of clones, migration, and invasion of A549 cells and the protein expression levels of IL-6, p-JAK2, p-STAT3, N-cadherin, and vimentin in the low-, medium-, and high-dose limonin groups significantly decreased, compared with those in the NC group, and the apoptosis rate and E-cadherin protein expression significantly increased (P < 0.05). The JAK2 activator coumermycin A1 attenuated the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and attenuated the apoptosis ability. The JAK2 inhibitor AG490 enhanced the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and enhanced the apoptosis ability. Conclusion Limonin can inhibit the malignant biological behavior of NSCLC cells, such as proliferation, migration, and invasion, by inhibiting the JAK2/STAT3 pathway.

Effect of Xidi Liangxue recipe on the proliferation and apoptosis of HaCaT cells through the lncRNA NEAT1/miR-485-5p/STAT3 regulatory network / 中华皮肤科杂志

Objective:To investigate the effect of Xidi Liangxue recipe on the proliferation and apoptosis of HaCaT cells through the long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) /microRNA (miR) -485-5p/signal transducer and activator of transcription 3 (STAT3) regulatory network. Methods:HaCaT cells were induced by interleukin-17 (IL-17), and the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3 was detected in IL-17-induced HaCaT cells and normal human epidermal keratinocytes (NHEK) by quantitative PCR (qPCR) and Western blot analysis, respectively. The location of lncRNA NEAT1 and miR-485-5p in IL-17-induced HaCaT cells was observed by fluorescence in situ hybridization (FISH), and the targeted regulatory relationship among lncRNA NEAT1, miR-485-5p and STAT3 was verified by double-luciferase reporter gene assay. Chinese herbs were decocted according to the Xidi Liangxue recipe, SD rats were divided into two groups to be gavaged with the above decoctions (medicated group) or physiological saline (control group) for 5 days, and then serum samples were collected from the above two groups of rats separately. The IL-17-induced HaCaT cells were divided into 4 groups: control group treated with the control sera, lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the control sera, Xidi Liangxue recipe group treated with the medicated sera, and Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the medicated sera. qPCR, Western blot analysis, flow cytometry, and cell counting kit (CCK8) assay were performed to determine the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3, and to evaluate cell proliferation and apoptosis. The two independent samples t-test was used for comparisons between two groups, one-way analysis of variance for comparisons among multiple groups, and least significant difference (LSD) t-test for multiple comparisons. Results:The IL-17-induced HaCaT cell group showed significantly increased relative expression levels of lncRNA NEAT1 and STAT3 mRNA (1.84 ± 0.21, 2.20 ± 0.24, respectively) and significantly increased protein expression of STAT3 and p-STAT3 (1.27 ± 0.13, 2.43 ± 0.16, respectively), but significantly decreased expression level of miR-485-5p (0.32 ± 0.04) compared with the NHEK group (lncRNA NEAT1 and STAT3 mRNA: 1.00 ± 0.11, 1.00 ± 0.11, respectively, both P < 0.05; STAT3 and p-STAT3 protein: 1.00 ± 0.11, 1.00 ± 0.10, t = 2.54, 3.02, respectively, both P < 0.05; miR-485-5p: 1.00 ± 0.12, t = 2.94, P = 0.015). FISH demonstrated that miR-485-5p and lncRNA NEAT1 were co-located in the cytoplasm of HaCaT cells. The double-luciferase reporter gene assay showed that the relative activity of luciferase was significantly lower in the miR-485-5p group than in the negative control group (both P < 0.05) after the transfection with wild-type lncRNA NEAT1 or STAT3 recombinant plasmids, while there were no significant differences between the miR-485-5p group and negative control group after the transfection with mutant lncRNA NEAT1 or STAT3 recombinant plasmids (both P > 0.05). Compared with the control group, the lncRNA-NEAT1 overexpression group showed significantly increased expression of lncRNA NEAT1 and STAT3 (including STAT3 mRNA, STAT3 protein, and p-STAT3 protein) in HaCaT cells (all P < 0.05), but significantly decreased miR-485-5p expression ( P < 0.05) ; the Xidi Liangxue recipe group showed significantly decreased expression of lncRNA NEAT1 and STAT3 (all P < 0.05), but significantly increased miR-485-5p expression compared with the control group ( P < 0.05) ; significantly decreased expression of lncRNA NEAT1 and STAT3, but significantly increased miR-485-5p expression was observed in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group compared with the lncRNA-NEAT1 overexpression group (all P < 0.05). After 24-, 48-, and 72-hour intervention, CCK8 assay showed that the proliferative activity of HaCaT cells was significantly higher in the lncRNA-NEAT1 overexpression group than in the control group (all P < 0.05), as well as in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group than in the Xidi Liangxue recipe group (all P < 0.05), and the cellular proliferative activity was significantly lower in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group and Xidi Liangxue recipe group than in the control group (all P < 0.05). The apoptosis rate was significantly lower in the lncRNA-NEAT1 overexpression group (5.84% ± 0.28%) than in the control group (14.75% ± 0.83%, LSD- t = 3.48, P = 0.002), but significantly higher in the Xidi Liangxue recipe group (35.72% ± 3.62%) than in the control group (LSD- t = 5.34, P = 0.001) ; the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group showed significantly increased apoptosis rate (27.64% ± 2.82%) compared with the lncRNA-NEAT1 overexpression group (LSD- t = 9.06, P < 0.001) . Conclusion:The Xidi Liangxue recipe could inhibit the proliferation of IL-17-induced HaCaT cells and promote their apoptosis, which may be related to the intervention in the lncRNA NEAT1/miR-485-5p/STAT3 regulatory network.

Correlation Between Vimentin and Hepatocellular Carcinoma and Intervention Effect of Jianpi Yiqi Prescription / 中国实验方剂学杂志

ObjectiveTo study the correlation between Vimentin and hepatocellular carcinoma （HCC） and the mechanism of Jianpi Yiqi prescription against HCC through Vimentin. MethodCorrelation between Vimentin and HCC was analyzed based on the cancer genome atlas（TCGA）， clinical proteomic tumor analysis consortium（CPTAC）， STRING， and Cytoscape. SD rats were randomized into normal group （normal saline， ig， once/day， 4 weeks）， model group （normal saline， ig， once/day， 4 weeks）， low-dose， medium-dose， and high-dose （5.25， 10.5， 21 g·kg-1， ig， once/day， 4 weeks） JianpiYiqi prescription groups， signal transducer and activator of transcription 3 （STAT3） inhibition group （C188-9， 4.5 mg·kg-1， ip， once/day， 4 weeks）， and glycoprotein 130 （gp130） inhibition group （SC144， 4.5 mg·kg-1， ip， once/day， 4 weeks）， 10 rats in each group. Diethylnitrosamine （DEN， 70 mg·kg-1 body weight， ip） was injected in rats except the normal group to induce HCC. After the modeling， administration began. After last administration， Real-time polymerase chain reaction（Real-time PCR） was performed to determine Vimentin mRNA level in rat liver tissue. Caspase-3 activity in liver tissue was detected by colorimetry， and expression of Rho kinase （ROCK）1， ROCK2， aurora kinase B （AURKB）， Zinc-finger protein 148 （ZNF148）/zinc-binding protein-89 （ZBP-89）， STAT3， p-STAT3， total Vimentin， and phosphorylated （p）-Vimentin in liver tissue and Vimentin in liver tissue nucleus detected by Western blot. Serum Vimentin concentration was measured by enzyme-linked immunosorbent assay （ELISA）. ResultVimentin mRNA level was high in tissues from HCC patients with different cancer stages （stage Ⅰ-Ⅳ）， different pathological grades （G1-G3）， no regional lymph node metastasis （N0）， and different subtypes （P<0.01）. Vimentin mRNA expression was higher in tissues from patients with lymph node metastasis than in patients without lymph node metastasis and normal samples. Vimentin protein level was decreased in HCC tissues （P<0.01）. Vimentin gene has 4 mutations which can induce change in the primary structure of Vimentin protein and patients with Vimentin gene mutation had short disease free survival time （P<0.01）. The mRNA expression of Vimentin was negatively associated with HCC cell purity （P<0.01） but was positively associated with the infiltration levels of cancer-associated fibroblasts， M2 macrophages， myeloid dendritic cell and other immune cells in tumor microenvironment （P<0.01）. Association analysis results showed that the expression of Vimentin was correlated with the STAT3 expression in HCC tissues （P<0.01）. As for the animal experiment， Vimentin mRNA level and protein levels of total Vimentin and p-Vimentin in liver tssue， Vimentin protein level in liver tissue nucleus， Vimentin in rat serum， ROCK2， AURKB， STAT3 and p-STAT3 in liver tissues were up-regulated （P<0.01） and protein level of negative regulator ZBP-89 was reduced in the model group （P<0.01） compared with those in the normal group. Activity of Caspase-3 in liver tissue increased and the ROCK1 protein level was increased in the model group compared with those in the normal group. STAT3 inhibitor， gp130 inhibitor， and medium-dose and high-dose Jianpi Yiqi prescription all can reduce the secretory Vimentin protein in serum， protein levels of total Vimentin and p-Vimentin in liver tissues， and Vimentin in liver tissue nucleus， and the protein levels of STAT3/Vimentin signaling pathway-related molecules， such as STAT3， p-STAT3， ROCK2， and AURKB and up-regulate the protein level of negative regulator ZBP-89 and activity of Caspase-3 （P<0.05， P<0.01）. Effect of medium-dose or high-dose Jianpi Yiqi prescription on Vimentin mRNA expression， STAT3 protein expression， ZBP-89 protein expression， ROCK2 protein expression， AURKB protein expression and Caspase-3 activity was not significantly different from that of STAT3 inhibitor. ConclusionVimentin， an important inflammatory molecule， is closely related to the occurrence and development of HCC and its expression， subcellular location and function may be affected by cancer-associated fibroblasts， M2 macrophages， myeloid dendritic cell， and IL-6/STAT3 signaling pathway， particularly by STAT3 molecule. Jianpi Yiqi prescription may exert therapeutic effect on HCC via regulating Vimentin through the STAT3/Vimentin signaling pathway.

Amentoflavone induces apoptosis and suppresses proliferation of thyroid carcinoma SW579 cells by inhibiting the activation of JAK2-STAT3 pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨阿曼托双黄酮（AF）对甲状腺癌SW579细胞中JAK2-STAT3通路活化及其细胞增殖和凋亡的影响。方法：用0、50、100、150、200 μmol/L的AF处理SW579细胞24、48、72 h，采用CCK-8和Celigo计数、FCM、WB及qPCR法检测AF对SW579细胞的增殖、凋亡、JAK2-STAT3通路活化及其下游调控基因c-Myc、Bcl2、survivin的mRNA及蛋白表达水平的影响。结果：AF处理后，SW579细胞增殖能力显著下降（P<0.05）且呈浓度依赖性，细胞凋亡呈浓度依赖性增多（P<0.05），细胞中JAK2-STAT3通路的活化受到显著抑制（P<0.05），其下游基因c-Myc、Bcl2、survivin的mRNA及蛋白表达均明显下降（均P<0.05）。结论：AF可通过抑制SW579细胞中JAK2-STAT3通路活化及其下游基因的表达而抑制SW579细胞的增殖并促进其凋亡，有望成为治疗甲状腺癌的有效药物。

Shaoyaotang Alleviates Ulcerative Colitis by Regulating Th17/Treg Balance Through IL-6/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the role of interleukin-6 （IL-6）/signal transducer and activator of transcription 3 （STAT3） pathway in the balance of T helper 17 （Th17）/regulatory T （Treg） cells in ulcerative colitis （UC） with internal dampness-heat accumulation syndrome and the intervention mechanism of Shaoyaotang. MethodA total of 60 SD rats were randomized into blank group （equivalent volume of normal saline）， model group （equivalent volume of normal saline）， western medicine control group （0.42 g·kg-1 mesalazine）， and low-dose （11.1 g·kg-1）， medium-dose （22.2 g·kg-1）， and high-dose （44.4 g·kg-1） Shaoyaotang groups. UC with internal dampness-heat accumulation syndrome was induced in rats with the compound method except for the blank group. The administration lasted 14 days for each group. At 24 h after the last administration， rats were killed and the spleen and colon tissues were separated. The histopathological changes of colon were observed based on hematoxylin and eosin （HE） staining and the levels of interleukin-17 （IL-17） and transforming growth factor-β1 （TGF-β1） in colon tissue were detected by immunohistochemistry （IHC）. Flow cytometry was employed to determine the levels of Th17/Treg cells in the spleen， and Western blot to measure the levels of IL-6 and STAT3 proteins in colon tissue. ResultCompared with the blank group， the model group had lesions such as congestion and erosion， low percentage of spleen Treg cells （P<0.01）， high percentage of Th17 cells （P<0.01）， and high levels of IL-6 and STAT3 proteins in colon tissue （P<0.01）. Compared with the model group， the administration groups showed alleviation of colon injury， high percentage of spleen Treg cells （P<0.05， P<0.01）， low percentage of Th17 cells （P<0.01）， and low levels of IL-6 and STAT3 proteins in colon tissue （P<0.01）. ConclusionShaoyaotang regulates the balance of Th17/Treg by inhibiting the IL-6/STAT3 pathway， thereby relieving the pathological damage of UC rats with internal dampness-heat accumulation syndrome and affecting their immune function.