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.

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。

Baricitinib improves pulmonary fibrosis in mice with rheumatoid arthritis-associated interstitial lung disease by inhibiting the Jak2/ Stat3 signaling pathway

Abstract Objective The study explored improvements in pulmonary inflammation and fibrosis in a bovine type II collagen-induced rheumatoid arthritis-associated interstitial lung disease mouse model after treatment with baricitinib and the possible mechanism of action. Methods A rheumatoid arthritis-associated interstitial lung disease mouse model was established, siRNA Jak2 and lentiviral vectors were transfected with human embryonic lung fibroblast cells. And the levels of relevant proteins in mouse lung tissue and human embryonic lung fibroblasts were detected by Western blotting. Results The levels of JAK2, p-JAK2, p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were increased in the lung tissues of model mice (P < 0.5) and decreased after baricitinib intervention (P < 0.05). The expression levels of p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were reduced after siRNA downregulation of the JAK2 gene (P < 0.01) and increased after lentiviral overexpression of the JAK2 gene (P < 0.01). Conclusion Baricitinib alleviated fibrosis in the lung tissue of rheumatoid arthritis-associated interstitial lung disease mice, and the mechanism of action may involve the downregulation of Smad3 expression via inhibition of the Jak2/Stat3 signaling pathway, with consequent inhibition of the profibrotic effect of transforming growth factor-β1.

KIF22 promotes the proliferation and glycolysis of melanoma by activating EGFR/STAT3 signaling

Abstract Objectives Melanoma is one of the leading causes of cancer death. Kinesin Family member 22 (KIF22) is essential for the invasion of melanoma cells, but the role and mechanism of KIF22 in the proliferation and glycolysis in melanoma remains unknown. Methods KIF22 expression in melanoma tissues and the relationship between KIF22 high expression and overall survival rate in patients with melanoma were analyzed using the Tnmplot database. KIF22 expression in melanoma cells was examined by western blot. Then, KIF22 was silenced and CCK-8 assay, EDU staining and flow cytometry analysis were adopted for assessing cell proliferation and apoptosis. In addition, the glycolysis metabolism of melanoma cells was reflected by detecting Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR). The expression of proteins related to apoptosis, glycolysis and EGFR/STAT3 signaling was tested by western blot. Subsequently, melanoma cells were treated with EGF or Colivelin to further elucidate the regulatory effect of KIF22 on EGFR/STAT3 signaling. Results KIF22 expression was notably upregulated in melanoma tissues and cells, and KIF22 high expression was associated with a poor prognosis. Moreover, KIF22 insufficiency suppressed proliferation and accelerated apoptosis of melanoma cells. Additionally, glycolysis was reduced by KIF22 depletion, evidenced by the decreased ECAR and increased OCR, accompanied by the downregulated expression of HK2, PKM2 and LDHA. Importantly, the impacts of KIF22 depletion on the progression of melanoma were partially attenuated after EGF or Colivelin treatment. Conclusion Collectively, KIF22 knockdown suppressed the proliferation and glycolysis and facilitated the apoptosis of melanoma cells by inactivating EGFR/STAT3 signaling.

Suppressing STAT3 activation impairs bone formation during maxillary expansion and relapse

Abstract Objectives The mid-palatal expansion technique is commonly used to correct maxillary constriction in dental clinics. However, there is a tendency for it to relapse, and the key molecules responsible for modulating bone formation remain elusive. Thus, this study aimed to investigate whether signal transducer and activator of transcription 3 (STAT3) activation contributes to osteoblast-mediated bone formation during palatal expansion and relapse. Methodology In total, 30 male Wistar rats were randomly allocated into Ctrl (control), E (expansion only), and E+Stattic (expansion plus STAT3-inhibitor, Stattic) groups. Micro-computed tomography, micromorphology staining, and immunohistochemistry of the mid-palatal suture were performed on days 7 and 14. In vitro cyclic tensile stress (10% magnitude, 0.5 Hz frequency, and 24 h duration) was applied to rat primary osteoblasts and Stattic was administered for STAT3 inhibition. The role of STAT3 in mechanical loading-induced osteoblasts was confirmed by alkaline phosphatase (ALP), alizarin red staining, and western blots. Results The E group showed greater arch width than the E+Stattic group after expansion. The differences between the two groups remained significant after relapse. We found active bone formation in the E group with increased expression of ALP, COL-I, and Runx2, although the expression of osteogenesis-related factors was downregulated in the E+stattic group. After STAT3 inhibition, expansive force-induced bone resorption was attenuated, as TRAP staining demonstrated. Furthermore, the administration of Stattic in vitro partially suppressed tensile stress-enhanced osteogenic markers in osteoblasts. Conclusions STAT3 inactivation reduced osteoblast-mediated bone formation during palatal expansion and post-expansion relapse, thus it may be a potential therapeutic target to treat force-induced bone formation.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis / 中国天然药物

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3βSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Bushen Huoxue Fang improves recurrent miscarriage in mice by down-regulating the JAK2/STAT3 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the efficacy of Bushen Huoxue Fang (BSHXF, a traditional Chinese medicine formula) for improving recurrent spontaneous abortion (RSA) in mice and the role of tyrosine kinase (JAK2) and transcriptional activator (STAT3) signaling pathway in its therapeutic mechanism.@*METHODS@#Female CBA/J mice were caged with male DBA/2 mice to establish RSA mouse models, which were randomly divided into model group, dydrogesterone group and BSHXF group, with the female mice caged with male BALB/c mice as the control group (n=6). From the first day of pregnancy, the mice were subjected to daily intragastric administration of BSHXF, dydrogesterone, or distilled water (in control and model groups) for 12 days. After the treatments, serum levels of antithrombin III (AT-III), activated protein C (APC), tissue plasminogen activator (t-PA), progesterone, human chorionic gonadotropin (HCG), and estradiol (E2) were detected in each group using ELISA. HE staining was used to observe the morphological changes of the endometrium of the mice. Western blotting was performed to determine the expressions of p-JAK2, p-Stat3 and Bcl-2 in the placenta of the mice.@*RESULTS@#Compared with the control mice, the mouse models of RSA showed a significantly increased embryo loss rate with decreased serum levels of AT-III, T-PA, progesterone, APC and HCG, increased placental expressions of p-JAK2, p-STAT3 and Bax, and decreased expression of Bcl-2 (P < 0.05). Treatments with BSHXF and dydrogesterone both increased serum levels of AT-III, t-PA and HCG in the mouse models; Serum APC level was significantly reduced in BSHXF group and serum progesterone level was significantly increased in dydrogesterone group (P < 0.05).@*CONCLUSION@#BSHXF can improve the prethrombotic state and inhibit cell apoptosis by downregulating the JAK2/STAT3 pathway to increase the pregnancy rate in mouse models of RSA.

Angiotensin converting enzyme 2 alleviates infectious bronchitis virus-induced cellular inflammation by suppressing IL-6/JAK2/STAT3 signaling pathway / 生物工程学报

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.

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.

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.

Mechanism of Hedysari Radix Polysaccharide on Diabetic Nephropathy in db/db Mice Based on JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Hedysari Radix polysaccharide （HRP） on the Janus kinase 2 （JAK2）/signal transducer and activator of transcription protein 3 （STAT3） signaling pathway in diabetic nephropathy db/db mice. MethodFifty db/db mice were randomly divided into model group， irbesartan group （irbesartan suspension， 22.75 mg·kg-1）， and high-， medium-， and low-dose HRP groups （HRP suspension， 200， 100， 50 mg·kg-1） according to the body weight， with 10 mice in each group. Another 10 C57BL/6 mice were assigned to the normal group. The mice were treated with corresponding drugs by gavage， while those in the normal group and the model group received distilled water at 5 mL·kg-1. The mice in the six groups were administered once a day by gavage for 12 consecutive weeks. The uric acid （UA）， triglycerides （TG）， and total cholesterol （TC） were detected. Periodic acid-Schiff （PAS） staining and Masson staining were used to observe the pathological changes in kidney tissues. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and mRNA expression levels of JAK2， STAT3， suppressor of cytokine signaling 3 （SOCS3）， and tumor necrosis factor-α （TNF-α） in the kidney. ResultAfter 12 weeks of treatment， compared with the normal group， the model group showed significant pathological ultrastructural changes in kidney tissues and increased UA， TG， and TC levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed improvement in pathological ultrastructure of kidney tissues and reduced UA， TG， and TC levels （P<0.05， P<0.01）. Compared with the normal group， the model group showed a decrease in SOCS3 protein and mRNA expression levels and an increase in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed an increase in SOCS3 protein and mRNA expression levels and a decrease in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.05， P<0.01）. ConclusionHRP can alleviate renal damage in diabetic nephropathy to a certain extent， and its mechanism may be related to the inhibition of the activation of the JAK2/STAT3 signaling pathway.

Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Qiling Baitouweng Tang （QLBTWT） on proliferation and apoptosis， Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） signaling pathway and interleukin-10 （IL-10） in diffuse large B-cell lymphoma （DLBCL）. MethodWith human DLBCL cells OCI-LY10 and U2932 as research objects， cell proliferation was detected by cell counting kit-8 （CCK-8） assay. After treatment with 0， 4.6， 9.3， 18.7， 37.5， 75， 150 mg·L-1 QLBTWT for 24 h， the half-inhibitory concentration （IC50） of OCL-LY10 and U2932 cells was calculated to be 9.33， 16.13 mg·L-1， respectively， based on which， 9.5， 19， 38 mg·L-1 QLBTWT were selected for subsequent experiments. After 0， 9.5， 19， 38 mg·L-1 QLBTWT treatment for 24 h， the zymogen activities of Caspase-3， Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits （colorimetric）， and the IL-10 expression was detected by enzyme-linked immuno sorbent assay （ELISA）. The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly adenosine diphosphate ribose polymerase （cleaved PARP）， cleaved Caspase-3］， JAK2， STAT3， phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3） pathway proteins， and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0， 9.5， 19， 38 mg·L-1 QLBTWT were all tested by Western blot. ResultAfter QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， cell proliferation was inhibited in each QLBTWT group compared with that in the control group （P<0.05， P<0.01）. The zymogens of Caspase-3， Caspase-8 and Caspase-9 were activated （P<0.01）， and there was an increase in cell apoptosis （P<0.05， P<0.01） and cell cycle arrest at Gap phase1 （G1） phase in 9.5， 19 and 38 mg·L-1 QLBTWT group （P<0.05， P<0.01）. After 9.5， 19 and 38 mg·L-1 QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， the expressions of Bcl-2， p-JAK2 and p-STAT3 proteins were decreased （P<0.01）， and the expressions of Bax， cleaved PARP and cleaved Caspase-3 proteins were increased （P<0.01）， but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group， the expressions of c-Myc， p-JAK2， and p-STAT3 proteins were down-regulated in 19 mg·L-1 QLBTWT group and 19 mg·L-1 QLBTWT+10 μg·L-1 IL-10 group （P<0.05， P<0.01）， and up-regulated in 10 μg·L-1 IL-10 group （P<0.05， P<0.01）， while there was no difference in JAK2/STAT3 proteins. ConclusionQLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932， and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

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.

PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy

Stroma surrounding the tumor cells plays crucial roles for tumor progression. However, little is known about the factors that maintain the symbiosis between stroma and tumor cells. In this study, we found that the transcriptional regulator-signal transducer and activator of transcription 3 (Stat3) was frequently activated in cancer-associated fibroblasts (CAFs), which was a potent facilitator of tumor malignancy, and formed forward feedback loop with platelet-activating factor receptor (PAFR) both in CAFs and tumor cells. Importantly, PAFR/Stat3 axis connected intercellular signaling crosstalk between CAFs and cancer cells and drove mutual transcriptional programming of these two types of cells. Two central Stat3-related cytokine signaling molecules-interleukin 6 (IL-6) and IL-11 played the critical role in the process of PAFR/Stat3 axis-mediated communication between tumor and CAFs. Pharmacological inhibition of PAFR and Stat3 activities effectively reduced tumor progression using CAFs/tumor co-culture xenograft model. Our study reveals that PAFR/Stat3 axis enhances the interaction between tumor and its associated stroma and suggests that targeting this axis can be an effective therapeutic strategy against tumor malignancy.

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.