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Objective To observe the effects of electroacupuncture on central inflammatory response and neurotransmitter release in rats with post stroke spasticity(PSS);To exploring the mechanism in treating PSS based on IL-6/JAK2/STAT3 signaling pathway.Methods Totally 30 male SD rats were randomly divided into sham-operation group,model group and electroacupuncture group,with 10 rats in each group.The PSS model was prepared by the method of suture and N-methyl-D-aspartic acid receptor injection into internal capsule.The rats in the electroacupuncture group were electroacupulated on the affected side of the body at"Quchi"and"Yanglingquan"for 30 min/d for consecutive 7 d.The sham-operation group and the model group were only fixed without any interventions.The Zea Longa neurological function score and the modified Ashworth muscle tension score were evaluated before and after treatment in each group;the pathological changes of the cortex on the ischemic side were observed by HE staining;the contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and γ-aminobutyric acid(GABA)in cortex on the ischemic side were detected by ELISA;the content of glutamate(Glu)was detected by biochemical kit;Western blot was used to detect the expressions of tyrosine kinase 2(JAK2),p-JAK2,signal transduction and transcription activating factor 3(STAT3)and p-STAT3 protein in ischemic cortex;RT-PCR was used to detect the mRNA expressions of JAK2 and STAT3 in ischemic cortex.Results Compared with the sham-operation group,neurological function score and muscle tension score significantly increased in the model group(P<0.01),with disorganized neurons in cerebral cortex,nucleus accumbens,the contents of IL-6,TNF-α and Glu significantly increased,the content of GABA significantly decreased(P<0.01),and p-JAK2,p-STAT3 proteins and JAK2 and STAT3 mRNA expression significantly increased(P<0.01,P<0.05).Compared with the model group,the neurological function score and muscle tension score of rats in the electroacupuncture group were significantly decreased(P<0.05),the degree of neuronal damage in cerebral cortex was reduced,the cell contour was clear,the content of IL-6,TNF-α and Glu were significantly decreased,and the content of GABA significantly increased(P<0.05,P<0.01),p-JAK2,p-STAT3 protein and JAK2,STAT3 mRNA expression significantly decreased(P<0.01).Conclusion Electroacupuncture may alleviate central inflammatory response and improve limb spasticity of PSS model rats by inhibiting the IL-6/JAK2/STAT3 signaling pathway.
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Objective To investigate the effect of folic acid–modified liposome quercetin (FLQ) on the proliferation and apoptosis of triple negative breast cancer (TNBC) cells and explore its underlying mechanism. Methods CCK-8 was used to detect the effect of FLQ on TNBC cell viability. Colony formation assay was conducted to detect the effect of FLQ on TNBC cell proliferation. Flow cytometry was performed to detect the effect of FLQ on TNBC cell apoptosis, the levels of intracellular ROS, and mitochondrial membrane potential. Western blot analysis was conducted to detect the expression levels of JAK2/STAT3 signaling pathway-related and apoptosis-related proteins. Results FLQ inhibited the proliferation and promoted the apoptosis of MDA-MB-231 cells (P=0.023, P<0.001). It promoted mitochondrial membrane potential collapse and increased the intracellular ROS levels of MDA-MB-231 cells (P=0.003, P=0.034); inhibited the phosphorylation levels of JAK2 and STAT3; upregulated the expression levels of the proapoptotic proteins Bax, Bak, cytochrome C, and Cleaved-Caspase-3 (P<0.001, P<0.001); and downregulated the expression levels of the antiapoptotic proteins Bcl2 and Bcl-xL (P=0.037, 0.028). Conclusion FLQ inhibits the proliferation and induces the apoptosis of MDA-MB-231 cells. These effects may be related to the activation of the mitochondrial apoptosis pathway through the inhibition of the JAK2/STAT3 signaling pathway.
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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.
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Purpose: Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). Methods: Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. Results: It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. Conclusions: NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.
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Doxorubicin , Cell Culture Techniques , STAT3 Transcription Factor , Cerebrum , NeuroblastomaABSTRACT
Objective To investigate the effects of Helicobacter pylori(Hp)on the proliferation,migration,apoptosis and inflammatory response of human umbilical vein endothelial cells(HUVEC)through activation of STAT3/nuclear factor κB(NF-κB)pathway.Methods HUVEC were divided into control group(without Hp infection)and Hp group(multiplicity of infection=25).Cell morphology was observed with inverted microscopy,proliferation was detected by CCK-8 assay and plate cloning assay,and the migration ability was examined by Transwell migration as-say and wound healing assay.Flow cytometry was used to detect the apoptotic rate.Real-time fluo-rescence quantitative PCR was employed to measure the mRNA expression of cytotoxin-associat-ed gene A(CagA),IL-6,IL-8,IL-1β and TNF-α.Western blotting was applied to determine the protein expression of Cyclin D1,proto-oncogene C-Myc,MMP-2,MMP-9,PCNA,Bax,Bcl-2 and STAT3/NF-κB signaling pathway.Results Hp infection resulted in suppressed proliferation and migration abilities,decreased protein levels of Cyclin D1,PCNA,C-Myc,MMP-2,MMP-9 and Bcl-2,elevated protein levels of Bax,p-STAT3/STAT3,p-NF-KB p65/NF-κB p65,raised apoptotic rate,and significantly increased mRNA levels of IL-6,IL-8,IL-1β and TNF-α(2.71±0.05 vs 1.06±0.41,1.42±0.02 vs 0.92±0.11,2.50±0.29 vs 1.00±0.10,5.34±0.57 vs 1.00±0.16;P<0.01)when compared with the control group.Conclusion Hp infection inhibits proliferation and migra-tion,and induces apoptosis and inflammatory response in HUVEC through activation of the STAT3/NF-κB pathway.
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AIM To investigate the effects of Ophiopogonis Root Decoction on bleomycin(BLM)-induced idiopathic pulmonary fibrosis(IPF)in mice and to explore its metabolic modulation of immunity.METHODS The IPF mouse model was constructed by tracheal drip injection of BLM,and the mice were randomly divided into the control group,the model group,the pirfenidone group(0.3 g/kg)and the high,medium and low dose groups of Ophiopogonis Root Decoction(18,9,4.5 g/kg).HE and Masson staining,ELISA,flow cytometry and immunohistochemistry were used to detect the histopathological changes of the lung,the levels of Collagen I,HYP and TGF-β1,the proportion of PD-1+ CD4+T cells in plasma,and the expressions of p-STAT3,PD-1,PD-L1 and IL-17A in lung tissue,respectively.RESULTS Compared with the control group,the model group displayed significantly higher level of lung coefficients(P<0.01),more severe pulmonary inflammatory cell infiltration and collagen fiber deposition,and increased pulmonary fibrosis score(P<0.01),increased levels of Collagen I,HYP and TGF-β1(P<0.01),increased proportion of PD-1+ CD4+ T cells in plasma(P<0.01),increased pulmonary expression of p-STAT3,PD-1,PD-L1 and IL-17A(P<0.01).Compared with the model group,the Ophiopogonis Root Decoction groups shared lower levels of lung coefficients(P<0.05),less pulmonary inflammatory cell infiltration and collagen fiber deposition,decreased pulmonary fibrosis score(P<0.05),decreased levels of Collagen I,HYP and TGF-β1(P<0.05),decreased proportion of PD-1+ CD4+T cells in plasma(P<0.05),and decreased pulmonary expression of p-STAT3,PD-1,PD-L1,and IL-17A(P<0.05).CONCLUSION Ophiopogonis Root Decoction can significantly reduce extracellular matrix(ECM)deposition and curb the progression of IPF via inhibition of STAT3/PD-1/PD-L1 immunomodulatory signaling pathway.
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Objective This study aims to investigate the regulatory effects of Fujiu Patch(composed of Sinapis Semen,Kansui Radix,Corydalis Rhizoma and Asari Radix et Rhizoma)on the CD4+ T helper 17 cell(Th17)/CD4+CD25+ regulatory T cell(Treg)balance in asthmatic rats via the signal pathway of IL-6/signal transducer and activator of transcription 3(STAT3)as well as IL-2/signal transducer and activator of transcription 5(STAT5),and to reveal its anti-asthma mechanisms.Methods An experimental asthma model was constructed by ovalbumin(OVA)combined with aluminum hydroxide sensitization and challenge,and then the rats were administered with Fujiu Patch at Dazhui(DU14),Feishu(BL13)and Shenshu(BL23)points for 4 hours each time,once every other day for 7 times.Immunohistochemistry was used to detect the positive expressions of Th17 specific cytokine(IL-17)and Treg transcription factor(Foxp3)in rat lung tissue.The percentage of Th17 and Treg cells in peripheral blood was examined by flow cytometry analysis,and the expressions of IL-6/STAT3 and IL-2/STAT5 pathway-related proteins in lung tissue were assayed with Western Blot.Results Compared to the model group,IL-17 positive expression in the rat lung showed a significant reduction in the Fujiu Patch group(P<0.01),while the positive expression of Foxp3 was obviously increased(P<0.05).Meanwhile,the protein expression levels of IL-6 and phospho-STAT3 were were significantly declined(P<0.01),and the protein expression levels of IL-2 and phospho-STAT5 were were significantly elevated(P<0.01).However,there was no significant alteration in the total protein expressions of STAT3 and STAT5(P>0.05).Furthermore,the proportion of Th17 cells in peripheral blood of rats in the Fujiu Patch group was lower than that in the model group,while the proportion of Treg cells was higher than that in the model group.Statistically-significant differences were observed(all P<0.01).Conclusion These findings indicate that Th17/Treg immune imbalance occurs in asthmatic rat.Fujiu Patch may exert anti-asthma effects via inhibiting the expression of IL-6,downregulating the expression of phospho-STAT3,diminishing the level of IL-17-producing Th17 cells,as well as increasing the expressions of IL-2-mediated STAT5 phosphorylation,raising the level of Foxp3-expressing Treg cells,promoting Th17/Treg balance and suppressing immune responses in rat with asthma.
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Objective To investigate the mechanism of morin-induced autophagy in non-small cell lung cancer A549 cells based on mTOR/STAT3 signaling axis.Methods A549 cells were divided into blank group and 30,60,90,120 and 150 μg·mL-1 of morin groups.After 24,48 and 72 hours of culture,the cell proliferation activity was detected by CCK-8 method,and the cell inhibition rate was calculated.A549 cells were divided into blank group and 30,90,150 μg·mL-1 morin groups.After 14 days of culture,the cell proliferation was detected by colony formation assay.After 24 hours of culture,the cell proliferation ability was detected by BeyoClickTM EdU-488.Apoptosis was detected by flow cytometry;acridine orange staining was used to detect cell autophagy;the formation of autophagosomes was observed by transmission electron microscopy.Western Blot was used to detect the expression levels of apoptosis,autophagy and mTOR/STAT3 signaling axis-related proteins in cells.A549 cells were divided into blank group,blank group + chloroquine(10 μg·mL-1)group,morin(30,150 μg·mL-1)group,morin(30,150 μg·mL-1)+ chloroquine(10 μg·mL-1)group.After 48 hours of intervention,the cell activity was detected by CCK-8 method,and the cell survival rate was calculated.Results Compared with the blank group,the inhibition rate of A549 cells in 60,90,120,150 μ g·mL-1 of morin group was significantly increased after 24 hours of intervention(P<0.05,P<0.001).The inhibition rates of A549 cells in 30,60,90,120 and 150 μg·mL-1 of morin groups were significantly increased after 48 and 72 hours of intervention(P<0.001).The number of A549 cell colonies and the number of green fluorescent proliferation positive cells in the 30,90,150 μg·mL-1 of morin groups were significantly decreased(P<0.01,P<0.001),the apoptosis rate was significantly increased(P<0.01,P<0.001),and the protein expression level of cleaved-PARP was significantly increased(P<0.001).The protein expression levels of p-P38/P38 MAPK in A549 cells of 90 and 150 μg·mL-1 of morin groups were significantly increased(P<0.01,P<0.001).Different degrees of orange fluorescence appeared in A549 cells of 30,90 and 150 μg·mL-1 of morin groups,and the orange fluorescence of 90 and 150 μg·mL-1 of morin groups was significant.Autophagosomes and autolysosomes appeared in the cytoplasm of A549 cells in 150 μg·mL-1 of morin group,respectively.The protein expression of LC3-Ⅱ in A549 cells of 150 μg·mL-1 of morin group was significantly up-regulated(P<0.05).The protein expression of Atg16L1-Ⅱ in A549 cells of 90,150 μg·mL-1 of morin group was significantly up-regulated(P<0.001),and the protein expressions of p-mTOR/mTOR and p-STAT3/STAT3 were significantly down-regulated(P<0.001).Compared with the morin(150 μg·mL-1)group,the survival rate of A549 cells in the morin(150 μg·mL-1)+chloroquine(10 μg·mL-1)group was significantly increased(P<0.05).Conclusion Morin can promote the apoptosis of A549 cells and induce autophagy in A549 cells,and the mechanism may be related to mTOR/STAT3 axis.
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【Objective】 To explore the expression of PCDH9 loss in regulating cell cycle and promoting tumor progression. 【Methods】 The clinical records of 127 cases of prostate cancer treated during 2018 and 2023 were collected, including 87 paraffin tissue samples from the G4-5 group and 40 from the G1-3 group. The expressions of PCDH9, p53, Rb and STAT3 were detected with immunohistochemical staining, and the relationship between their expressions and clinicopathological characteristics was analyzed. 【Results】 The expression deletion rate of PCDH9 in prostate cancer tissues in G4-5 group (44.8% vs.7.5%) was significantly higher than that in G1-3 group (P<0.001). The positive expression rates of p53 and STAT3 were 34.5% and 89.7%, respectively, and the expression loss rate of Rb was 27.6% in G4-5 group. The expression loss rates of PCDH9 and Rb were associated with neuroendocrine-like histological morphology, nerve invasion and vascular invasion (P<0.05). In G4-5 group of prostate cancer, PCDH9 expression was positively correlated with the expressions of p53 (r=0.345, P<0.05), Rb (r=0.503, P<0.05) and STAT3 (r=0.224, P<0.05). 【Conclusion】 PCDH9 is prone to loss of expression in high-group prostate cancer tissues, especially in cases with neuroendocrine-like histological morphology, which may regulate the cell cycle through the STAT3 signaling pathway, thereby promoting tumor progression.
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Objective @#To study the effect and mechanism of high glucose on mesothelial-mesenchymal transition (MMT) of peritoneal mesothelial cells (HMrSV5) , and the protective effect of pharmacological blocking of signal transducer and activator of transcription 3 (STAT3) on rat peritoneal fibrosis (PF) model . @*Methods @#The animals were divided into three groups : the sham group , the model group , and the STAT3 inhibitor group . A miniature per- itoneal dialysis catheter was implanted under the dorsal skin of rat and the rat peritoneal fibrosis model was induced by daily injection of high glucose dialysate . After 10 weeks , HE staining was used to evaluate the histology of the peritoneum , and the level of transforming growth factor-β1 (TGF-β1) in the peritoneum was measured by immuno- histochemistry . HMrSV5 was cultured in high glucose and the optimal stimulation concentration of high glucose was determined by Western blot. High glucose was used to stimulate HMrSV5 after successful transfection with si - STAT3 and Western blot was used to measure the protein level of STAT3 , p-STAT3 , and the key enzymes of glycol- ysis 6-phosphofructo-2-kinase/fructose-2 , 6-biphosphatase 3 (PFKFB3) and lactate dehydrogenase A (LDHA) .@*Results @#HE staining showed that administration of STAT3 inhibitor ( BP-1-102) could inhibit the thickening of subperitoneal tissue and the proliferation of vessels in HG dialysis rats . The expression of TGF-β1 in the rats perito- neum of the model group was significantly higher than that in the sham group , and the level of TGF-β1 was marked- ly lower in the STAT3 inhibitor group compared to the model group (P < 0. 05) . Compared to the control group , high glucose induced the up-regulation of α-smooth muscle actin ( α-SMA) , the down-regulation of E-cadherin and STAT3 activation in HMrSV5 (P < 0. 05) . Mesothelial cells treated with high glucose also exhibited high expres- sion of the key enzymes of glycolysis ( PFKFB3 , LDHA) ( P < 0. 05) , and si-STAT3 can effectively inhibit the overexpression of PFKFB3 and LDHA induced by high glucose ( P < 0. 05) . @*Conclusion @#STAT3 is involved in high glucose-induced HMrSV5 hyperglycolysis and MMT , and targeting STAT3 alleviates peritoneal fibrosis and an- giogenesis during peritoneal dialysis treatment in rats .
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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.
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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.
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@#[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。
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@#[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。
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ObjectiveTo investigate the mechanism of Baitouweng Tang in inhibiting the growth of esophageal cancer (EC) cells by regulating budding uninhibited by benzimidazoles 1 (BUB1)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. MethodGene chip technology was used to explore the differential gene expression between esophageal cancer tissues and normal tissues and identified differentially expressed genes. The differentially expressed genes were analyzed by bioinformatics methods. EC cells were treated with 25, 50, 100, 200, 400, 800 mg·L-1 Baitouweng Tang. EC cell viability was detected by Thiazolyl Blue (MTT) colorimetry. Cell cycle and apoptosis were measured by flow cytometry. The expression of BUB1 was measured by real time quantitative polymerase chain reaction (Real-time PCR). The protein levels of BUB1, STAT3, phosphorylated (p)-STAT3, Cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1), B-cell lymphoma-2 (Bcl-2), cysteinyl aspartate-specific proteinase(Caspase)-3, and Caspase-9 were measured by Western blot. The migration and invasion abilities of the cells were measured by wound-healing and Transwell invasion assays. ResultDifferentially expressed genes were primarily involved in biological processes, signaling pathways, and network construction related to cell mitosis, with BUB1 identified as a key core gene. Compared with the control group, Baitouweng Tang inhibited BUB1 expression (P<0.05,P<0.01). In vitro experiments showed that compared with the control group, Baitouweng Tang could significantly inhibit the growth (P<0.05,P<0.01), migration and invasion (P<0.05,P<0.01) of EC cells, induce apoptosis (P<0.05,P<0.01), and cause G2/M phase increase (P<0.01). After treatment with Baitouweng Tang, compared with the results in the control group, the expression of Caspase-3, and Caspase-9 in EC cells increased significantly (P<0.05,P<0.01), while the expression of Bcl-2, BUB1, CCNB1, and CDK1 decreased significantly (P<0.05,P<0.01). Moreover, the STAT3 signaling pathway was also found to play an important role in this process. ConclusionBaitouweng Tang may inhibit the growth of EC cells by downregulating BUB1 and mediating the STAT3 signaling pathway.
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ObjectiveTo explore the role of saikosaponin D (SSD) targeting signal transducer and activator of transcription 3 (STAT3) in inducing apoptosis of bladder cancer cells by computer-aided drug design and experimental verification. MethodThe druggability and biotoxicity of SSD were explored by Bayesian classifier modeling. The information about SSD, the active ingredient of Bupleuri Radix, was searched against the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP). The targets of SSD were predicted by PubChem, TCMSP, a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM), Coremine, an Encyclopedia of Traditional Chinese Medicine (ETCM), and SwissTargetPrediction. GeneCards, Therapeutic Target Database (TTD), and Online Mendelian Inheritance in Man (OMIM) were employed to predict the potential therapeutic targets of bladder cancer. Then, the common targets shared by SSD and bladder cancer were selected for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Molecular docking was adopted to explore the binding affinity and structural stability of SSD with target proteins. Cytoscape 3.9.1 was used to construct the STAT3-drug regulatory network and STAT3-apoptosis regulatory network. UM-UC-3 cells were treated with 0, 5, 10, 15 μmol·L-1 SSD for 24 h. Then, flow cytometry was used to detect the apoptosis of bladder cancer cells, and Western blot was employed to determine the protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter (Bad), STAT3, and phosphorylation (p)-STAT3. ResultBayesian classifier modeling and molecular docking showed that SSD had low biotoxicity and bound well to the target protein STAT3 to form a stable protein-ligand complex. There were 282 common targets between bladder cancer and SSD, among which STAT3 was the most central target. The GO enrichment analysis showed that the potential core therapeutic targets involved 3 036 biological processes, 82 cellular components, and 171 molecular functions. The KEGG enrichment analysis showed that the potential core targets were mainly related to the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and cell apoptosis pathway. The STAT3-drug regulatory network and STAT3-apoptosis regulatory network showed that 29 drugs interacted with STAT3, and 27 apoptosis-related genes had a strong correlation with STAT3. Flow cytometry showed that the apoptosis rate increased with the increase in SSD concentration (P<0.05). Western blotting results showed that SSD down-regulated the protein levels of p-STAT3 and Bcl-2 and up-regulated the protein levels of Bax and Bad in a concentration-dependent manner (P<0.05). ConclusionSSD has good druggability and low biotoxicity. It may promote the apoptosis of bladder cancer cells by targeting STAT3.
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@#Abstract: Signal transducer and activator of transcription 3 (STAT3) is an intracellular signaling factor that plays a critical role in various cellular processes, including the growth, differentiation, apoptosis, and immune response of cells. Aberrant activation of T helper cell 17 (Th17) is closely associated with the morbidity and progress of various autoimmune diseases. STAT3 participates in the pathogenesis of Sjögren syndrome by inducing excessive proliferation and abnormal differentiation of Th17 cells and affecting lymphocyte infiltration into exocrine glands. Therefore, targeting the STAT3 signaling pathway represents a potential novel therapeutic approach for the treatment of Sjögren syndrome. This review summarizes the research of STAT3 in the pathogenesis and progression of Sjögren syndrome through regulating Th17 cells, focusing on current inhibitors targeting the STAT3 signaling pathway as potential therapeutic targets for Sjögren syndrome.
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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.
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Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.
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ObjectiveThe differential expression of microRNAs (miRNAs) between the active stage and the remission stage of ulcerative colitis (UC) was analyzed by bioinformatics method, and the regulatory relationship was constructed by screening the differentially expressed genes (DEGs). The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database (GEO), and the most differentially expressed miRNAs were screened by GEO2R, Excel, and other tools as research objects. TargetScan, miRTarbase, miRDB, STRING, TRRUST, and Matescape databases were used to screen key DEGs, predict downstream transcription factors (TFs), gene ontology (GO), and conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments, the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate (DSS). Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days, and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor (SOCS1), phosphorylated transcriptional signal transductor and activator 3 (p-STAT3), phosphorylated Janus kinase 2 (p-JAK2), and retinoic acid-associated orphan receptor-γt (ROR-γt). The expression levels of transforming growth factor-β (TGF-β), interleukin-17 (IL-17), interleukin-6 (IL-6), and interleukin-10 (IL-10) in serum were detected by enzyme linked immunosorbent assay (ELISA). ResultThe GSE48957 dataset was screened from the GEO database, and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation, as well as signaling pathways such as stem cell signaling pathway, IL-17 signaling pathway, and helper T cell 17 (Th17) cell differentiation. The Matescape database was used to screen out 10 key DEGs, among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3, p-JAK2, and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice, up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10, increase the level of anti-inflammatory factors, and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice, inhibit the phosphorylation of STAT3, inhibit the differentiation of CD4+ T cells into Th17 cells, reduce the levels of pro-inflammatory factors (IL-17 and IL-6), and increase the levels of anti-inflammatory factors (TGF-β and IL-10). As a result, the inflammation of colon mucosa in UC mice was alleviated.