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.

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。

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.

Effect of Scutellarin on Proliferation of Acute Myeloid Leukemia Cells and Its Related Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of scutellarin (SCU) on proliferation, cell cycle and apoptosis of acute myeloid leukemia (AML) cells and its related molecular mechanism.@*METHODS@#Human AML HL-60 cells were cultured in vitro. The cells were treated with SCU at the concentration of 0, 2, 4, 8, 16, 32, 64 μmol/L, and the inhibition rate of cell proliferation was detected by CCK-8 method. Then HL-60 cells were treated with SCU at the concentration of 4, 8, 16 μmol/L, and the negative control group (NC group) was set. The cell cycle distribution and apoptosis were detected by flow cytometry, and the expression of cell cycle, apoptosis and JAK2/STAT3 pathway related proteins were detected by Western blot.@*RESULTS@#SCU significantly inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner(r =0.958,r =0.971). Compared with NC group, the proportion of cells in G0/G1 phase and apoptosis rate of HL-60 cells in 4, 8, 16 μmol/L SCU group were significantly increased, and the proportion of cells in S phase was significantly decreased (P <0.05). The relative protein expression levels of p21, p53, caspase-3 and Bax were significantly increased, while the relative protein expression levels of CDK2, cyclin E and Bcl-2 were significantly decreased (P <0.05). The ratio of p-JAK2/JAK2 and p-STAT3/STAT3 were significantly decreased (P <0.05). The changes of above-mentioned indexes were concentration dependent.@*CONCLUSION@#SCU can inhibit the proliferation of AML cells, induce cell cycle arrest and apoptosis, and its mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway / 中国中药杂志

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

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.

Inhibitory effect and molecular mechanism of sinomenine on human hepatocellular carcinoma HepG2 and SK-HEP-1 cells / 中国中药杂志

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.

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.

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.

ZNF460 Mediates STAT3 Phosphorylation to Promote Progression of Hepatocellular Carcinoma by Activating ZDHHC7 Transcription / 胃肠病学

Background: ZNF460 is a member of the zinc finger protein family transcription factors, and is involved in the regulation of a variety of cellular functions. It has been demonstrated to be closely related to digestive system cancers. Aims: To analyze the expression level of ZNF460 in hepatocellular carcinoma (HCC), and explore the effect and potential mechanisms of ZNF460 on tumor cell proliferation. Methods: Immunohistochemical staining was used to determine the expression level of ZNF460 in tissue microarray of 76 HCC and paired adjacent tissues, and the correlation between ZNF460 expression and TNM staging was analyzed. The effect of ZNF460 on HCC cell proliferation was evaluated by CCK‑ 8 and colony formation assays. Genes positively related to ZNF460 expression in HCC were screened through LinkedOmics database, and the KEGG and GSEA pathway enrichment analyses were performed; the possible downstream molecules of ZNF460 were explored and verified by overexpression or knockdown of ZNF460 in HCC cells combined with luciferase reporter assay and other experiments. Results: ZNF460 was highly expressed in HCC and was positively correlated with TNM staging. ZNF460 promoted the proliferation of HCC cells, and the underlying mechanism might be associated with the transcriptional activation of ZDHHC7, the coding gene of palmitoyl transferase DHHC7 and the subsequent STAT3 palmitoylation and phosphorylation. Conclusions: ZNF460 is highly expressed in HCC and promotes cell proliferation and tumor progression via STAT3 activation. It might be a promising molecular marker and therapeutic target for HCC.

Interleukin-6 in Skeletal Muscle Mass Regulation / 中国生物化学与分子生物学报

Interleukin-6 (IL-6) is a spreading pleiotropic cytokine, with both anti-inflammatory and proinflammatory effects. It not only participates in the body immune responses but also is involved in the biological regulative processes among different organs, tissues, and cells. IL-6 has both anti-inflammatory and pro-inflammatory effects. In the early stage of pathogen infection, IL-6 plays an anti-inflammatory role in the body, and its level is moderately increased in the body to resist inflammation and maintain internal homeostasis. However, a large amount of IL-6 release can cause excessive inflammation and trigger other pathological changes in the body. Il-6 also has the dual effect of stimulating the synthesis and degradation of skeletal muscle protein in regulating skeletal muscle mass. As an important locomotive organ, skeletal muscle is also one of the key targets of IL-6. IL-6 takes part in the biological control of skeletal muscle hypertrophy through regulating muscle satellite cell proliferation and differentiation under specific stresses. In addition IL-6 is also associated with skeletal muscle atrophy induced by aging and other pathological stresses. In addition, during exercise stress, skeletal muscle can also serve as an endocrine organ to secrete and release IL-6 that facilitates the "crosstalk" between skeletal muscle and other organs or tissues. As IL-6 plays as a versatile role in our body, this paper reviews the research progress of the mechanism of IL-6 in the regulation of skeletal muscle mass, which may provide theoretical support for revealing the molecular mechanism of skeletal muscle stresses and adaptations.

Mechanisms of Radix Tetrastigma on anti rheumatoid arthritis via regulating the balance of Th17/Treg / 中国临床药理学与治疗学

AIM: To explore the possible mechanism of Radix Tetrastigma (RT) on anti-rheumatoid arthritis (RA). METHODS: The rat model of RA was established by intradermal injection of complete Freund]s adjuvant into the right hind foot of SD rats. RT Extract with different dosage was continuous intragastric administration for 3 weeks, then, the degree of foot swelling, arthritis index score, joint heat and grip of each rat was measured respectively. ELISA was used to detect the expression levels of Interleukin (IL) 6, IL-17, IL-10, tumor necrosis factor-α, and rheumatoid factor. Fully automatic hemorheometer was applied to measure hemorheology indexes. The number of CD4

Effects of ethanolic extracts of Euonymus alatus on CCl / 中国药理学通报

Aim To explore the mechanism of ethanolic extracts of euonymus alatus on CCl4-induced hepatic fibrosis in mice by regulating JAK2/STAT3 signaling pathway. Methods Sixty C57BL/6J mice were randomly divided into control group,model group,EAL,EAM),EAH,and Silybin(n=10). Except for the control group,mice in other groups were injected with 25% CCl4 of 1.6 mL·kg

Mechanisms of Euphorbia fischeriana steud. alcohol extracts intreatment of HCC based on network pharmacology and experiment verification / 中国药理学通报

Aim To investigate the molecular mechanisms of alcohol extracts of Euphorbia fischeriana steud. against hepatocellular carcinoma (HCC) through a combination of network pharmacology analysis and experimental validation. Methods The active ingredients and targets of alcohol extracts of Euphorbia fischeriana steud. were determined through TCMSP, Swiss ADME, Swiss Target Prediction database and references. The databases DisGeNET and GeneCards were employed to screen potential HCC-related genes. Venny platform, STRING platform and Cytoscape software were applied to construct active ingredient-target-disease and protein-protein interaction (PPI) network maps. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed using the DAVID database. To assess the effects of Euphorbia fischeriana steud. alcohol extracts on BEL-7402 cells, the proliferation and apoptosis were detected by CCK-8, EdU and flow cytometry assays, and the related protein levels of JAK2/STAT3 pathway were analyzed by Western blot. Additionally, H22 hepatocellular carcinoma mouse model was used to evaluate the in vivo efficacy of Euphorbia fischeriana steud. alcohol extracts. Results A total of 916 HCC targeted genes, 30 active ingredients containing the related 567 potential targeted genes, and 115 intersection targets of disease and compounds were obtained. KEGG enrichment analysis identified JAK2/STAT3 signaling as a critical pathway. In vitro experiments showed the alcohol extracts of Euphorbia fischeriana steud. could inhibit proliferation, promote apoptosis and suppress JAK2/STAT3 signaling pathway in a dose-dependent manner in BEL-7402 cells. In addition, the alcohol extracts of Euphorbia fischeriana steud., either alone or in combination with sorafenib, dramatically blocked tumor growth in in vivo tests. Conclusions Euphorbia fischeriana steud. alcohol extracts have anti-cancer effects in HCC, and the molecular mechanisms may be connected to the regulation of JAK2/STAT3 signaling pathway.

Mechanism of Modified Danggui Shaoyao San in treatment of chronic atrophic gastritis based on network pharmacology and experimental verification / 中国药理学通报

Aim To predict the targets of Modified Danggui Shaoyao San ( MDSS) in the treatment of chronic atrophic gastritis ( CAG) based on network pharmacology and vertify the results based on experim-ention. Methods TCMSP, SWISS TARGETS, GENE CARDS and OMIM databases were used to screen the therapeutic targets of MDSS for CAG. STRING database and Cytoscape software were used to construct the protein interaction network and screen the core targets. Metascape database was used for GO analysis and KEGG enrichment pathways. And molecular docking was used for target validation. CAG rat model was pre¬pared by N-methyl-N'-nitroso-N-nitroguanidine free drink combined with sodium salicylate gastric lavage. The pathology of rat gastric mucosa was observed by hematoxylin-eosin staining,and the ultrastructure of ep¬ithelial cells was observed by transmission electron mi-croscopy. The serum IL-6 and IL-10 content was detected by enzyme-linked immunosorbent assay, and the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in rats was detected by qPCR and Western blot. Results MDSS acted on 189 targets, mainly involved in response to oxidative stress and apoptotic signaling pathway. KEGG analysis related to pathways in cancer and JAK-STAT signaling pathway. The experimental results showed that the MDSS could improve the degree of atrophy of gastric mucosa in CAG rats and improve the status of epithelial cells, down-regulate the serum IL-6 content of CAG rats, up-regulate the IL-10 content, and reduce the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in gastric mucosa with statistical significance. Conclusions MDSS treats CAG through multiple active ingredients, targets, and pathways, the mechanism of which may be related to the inhibition of the JAK2/STAT3 signaling pathway.