Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis. Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals. After 4 weeks, OVA atomization and gavage were used for stimulation and treatment. Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74, 3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline. HE, PAS and Masson staining were used to observe the lung histopathological changes in rats. The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA. The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue. Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious. The expressions of IL-6, IL- 23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01);Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased (P< 0.01). The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01). Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

To explore the mechanism of Dahuang Lingxian Formula in relieving inflammatory response of bile duct cells based on IL-6/JAK/STAT3 signaling pathway

Objective:To explore the mechanism of action of Dahuang Lingxian Formula in alleviating the inflammatory response of bile duct cells in LPS-induced intrahepatic bile duct inflammation model rats based on IL-6/JAK/STAT3 signaling pathway.Methods:Fifty SD rats were randomly divided into five groups,blank group,model group,choling tablets(0.5 g/kg),and low and high concentration groups(2.4 g/kg and 4.8 g/kg)of Dahuang Lingxian Formula,ten rats in each group.Except for the blank group,the rats in each group were injected with 1.25 mg/kg LPS at the common bile duct at one time to construct an animal model of intrahepatic bile duct infection.After gavage on day 8,liver tissues were taken from rats at the hepatic hilum,and the histopathological changes of the hepatic hilum and biliary tree were observed by HE staining.The expression levels of serum glutamic alanine transaminase(ALT),glutamic oxalacetic transaminase(AST),malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by biochemical method.The expression levels of interleukin 6(IL-6),Janus protein tyrosine kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3)in rat serum were measured by enzyme-linked immunosorbent assay(ELISA).Protein immunoblotting(WB)and real-time fluorescence quantitative PCR(RT-qPCR)were used to detect the expression levels of IL-6,JAK2,STAT3 protein and mRNA in biliary tree tissues.Results:①Compared with the blank group,the structures such as interlobular bile ducts in the hepatic sinusoids and portal duct area of the model rats were destroyed,and inflammatory cells infiltrated around them.The expression of ALT,AST,MDA,IL-6,JAK2 and STAT3 in the serum increased significantly,the expression level of SOD decreased,and the expression levels of IL-6,JAK2 and STAT3 proteins and mRNA increased.②Compared with the model group,the degree of liver pathological damage in rats in the Chiling Ning tablet group and the low and high concentration groups of Dahuang Lingxian Formula were improved,which could significantly reduce the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and up-regulate SOD in serum,and down-regulate the expression of IL-6,JAK2,STAT3 protein and mRNA,with the best effect in the high concentration group of Dahuang Lingxian Formula.③Compared with the choling tablet group,the rats in the low and high concentration groups of Dahuang Lingxian Formula tended to normalize the degree of liver pathological damage,without obvious inflammatory cell infiltration,and the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and the expression levels of IL-6,JAK2,STAT3 protein and mRNA in serum were reduced,and the expression levels of SOD were increased,with the best effect of Dahuang Lingxian Formula The treatment effect was best in the high concentration group.Conclusion:The mechanism may be related to the down-regulation of IL-6/JAK/STAT3 signaling pathway activation,and the best therapeutic effect was achieved by the high concentration group of Dahuang Lingxian Formula.

IL-37b suppresses epithelial mesenchymal transition in hepatocellular carcinoma by inhibiting IL-6/STAT3 signaling

Background: Interleukin-37 b(IL-37 b), a vital negative regulator of the innate immune system, has been reported to be a tumor inhibitor in different type of cancers. However, little is known about the relationship between IL-37 b and hepatocellular carcinoma(HCC). The present study aimed to investigate the potential roles of IL-37 b in HCC progression. Methods: Subjects( n = 237) were recruited, and serum IL-37 b was measured using ELISA. The tumorsuppressive capacity and underlying mechanisms of IL-37 b in HCC were investigated in vitro and in vivo. Results: Compared to healthy controls, serum IL-37 b levels were elevated in chronic hepatitis B(CHB) patients but decreased significantly in HBV-HCC patients, especially for those with portal venous tumor thrombus. Low level serum IL-37 b in HBV-HCC patients correlated with high HCC stage and poor overall survival and disease-free survival. In vitro and in vivo, recombinant human IL-37 b inhibited proliferation and metastasis in HCC cells. Furthermore, IL-37 b inhibited epithelial mesenchymal transition in HCC cells in vitro by downregulating IL-6, pSTAT3(Y705), N-cadherin, and vimentin expression and by upregulating E-cadherin expression. These effects were partially reversed by transfection of adenovirus encoding human IL-6. Conclusions: IL-37 b inhibits HCC growth, metastasis and epithelial mesenchymal transition by regulating IL-6/STAT3 signaling. Serum IL-37 b may be a biomarker for HBV-HCC and its staging.

KIAA1199 induces advanced biological behavior and development of ovarian cancer through activation of the IL-6/STAT3 pathway

Recently,abnormal expression of KIAA1199 has been detected in Epithelial Ovarian Cancer(EOC).However,the underlined anti-ovarian cancer mechanism of KIAA1199 remains to be enlightened.In our study,we performed to elucidate the effects of KIAA1199 on the advanced biological behavior of EOC cells through activation of the IL-6/STAT3 pathway.Confirmed by immunohistochemistry,KIAA1199 was highly expressed in ovarian borderline and malignant epithelial tumors.A retrospective analysis found that EOC patients with low expression of KIAA1199 had a significantly higher 5-year survival rate than those with high expression.Mechanistically,IL-6 was used to stimulate EOC cells,and the expression of KIAA1199,STAT3 and p-STAT3 increased after IL-6 stimulation.These results could show that KIAA1199 is transcriptionally activated by IL6/STAT3 pathway,thereby accelerating the deterioration of EOC.KIAA1199 could also be used as a poor prognosis factor and potential target in treatment.

INHIBITION OF IL-6-INDUCED STAT3 ACTIVATION IN MYELOMA CELLS BY PROTEIN KINASE A

Objective: To investigate the regulation effect of protein kinase A on IL-6-induced STAT3 activation in myeloma cells. Methods: Two human myeloma cell lines-Sko-007 and U266 were pretreated with Forskolin, a protein kinase A antagonist, and then stimulated by IL-6. The activation state of STAT3 in these two cells were examined by electrophoretic mobility shift assay (EMSA). Results: Although PKA pathway itself doesn’t participate in IL-6 signal transduction in Sko-007 and U266 cells, activation of protein kinase A can inhibit IL-6-induced STAT3 activation in these two cell lines. Conclusion: There exists an inhibitory effect of protein kinase A on STAT3 activation in human myeloma cells treated by IL-6.

YD383、YD439对JAK-STAT6信号传导通路作用的特异性研究

目的:探讨化合物YD383和YD439对STAT6信号传导通路的作用是否具有特异性。方法:通过瞬时转染的方法将分别依赖于STAT1和STAT6活性的带有荧光素酶报告基因的质粒转染至HeLa细胞株,两种细胞株在各自相应的细胞因子(IFN-γ或IL-4)刺激下激活JAK-STAT1或JAK-STAT6信号传导通路。通过测定荧光素酶的活性来评价细胞株在化合物存在的情况下报告基因的表达。结果:YD383和YD439均能降低相应细胞因子刺激引起的报告基因的表达,并且具有剂量依赖性。随着化合物剂量的增加,报告基因的表达降低(P<0.05)。但化合物引起的两种转染细胞报告基因表达降低的差异比较无统计学意义(P>0.05)。结论:两种化合物对JAK-STAT1和JAK-STAT6信号传导通路均有抑制作用,对JAK-STAT6通路的抑制不具有特异性。

The Influence of Gastrodin on Expression of IL-10, STAT3 and JAK2 in Epileptic Rats’ Hippocampus

Objective: To explore the influence of gastrodin on IL-10, JAK2 and STAT3 in hippocampus of epileptic rats induced by pentylenetetrazol and the role of the IL-10 pathway in epilepsy. Methods: 50 adult male Wistar rats were randomly divided into 5 groups: normal control group (NC Group), epilepsy model group (EP Group), low doses of gastrodin + EP Group (GE1 Group), medium doses of gastrodin + EP Group (GE2 Group), high doses of gastrodin + EP Group (GE3 Group). EP group and GE Groups were injected subthreshold doses of pentylenetetrazole (PTZ) by intraperitoneal once a day until reaching the ignited standards. GE groups were respectively injected 4, 6, 8 mg/kg gastrodin by intraperitoneal. All groups were administered for 28 consecutive days. The behavioral changes of the rats were observed and recorded daily 1 hour after the injection. mRNAs of IL-10, STAT3 and JAK2 in hippocampus were measured by RT-qPCR, and proteins by Western blot. Results: Compared GE2 group with EP group, the incubation of seizure was significantly prolonged (P Conclusions: Gastrodin can increase the expression of IL-10, and reduce the expression of STAT3 and JAK2, which may play an antiepileptic effect through regulating JAK2/STAT3 signaling pathways by IL-10.

Sphingosine kinase 1 promotes growth of glioblastoma by increasing inflammation mediated by the NF-κB/IL-6/STAT3 and JNK/PTX3 pathways

Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence rate.Currently,mechanisms of GBM are still unclear and there is no effective drug for GBM in the clinic.Therefore,it is urgent to identify new drug targets and corresponding drugs for GBM.In this study,in silico analyses and experimental data show that sphingosine kinase 1(SPHK1)is up-regulated in GBM patients,and is strongly correlated with poor prognosis and reduced overall survival.Overexpression of SPHK1 promoted the proliferation,invasion,metastasis,and clonogenicity of GBM cells,while silencing SPHK1 had the opposite effect.SPHK1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK,activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of PTX3.SPHK1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression,promote inflammation and GBM growth.Inhibition of SPHK1 by the inhibitor,PF543,also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse models.In summary,we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM,which may provide opportunities for SPHK1-targeted therapy.

Myeloid peroxisome proliferator-activated receptor α deficiency accelerates liver regeneration via IL-6/STAT3 pathway after 2/3 partial hepatectomy in mice

Background:Liver regeneration is a fundamental process for sustained body homeostasis and liver function recovery after injury.Emerging evidence demonstrates that myeloid cells play a critical role in liver regeneration by secreting cytokines and growth factors.Peroxisome proliferator-activated receptorα(PPARα),the target of clinical lipid-lowering fibrate drugs,regulates cell metabolism,proliferation,and survival.However,the role of myeloid PPARαin partial hepatectomy(PHx)-induced liver regeneration remains unknown.Methods:Myeloid-specific PPARa-deficient(Ppara^(Mye−/−))mice and the littermate controls(Ppara^(fl/fl))were subjected to sham or 2/3 PHx to induce liver regeneration.Hepatocyte proliferation and mitosis were assessed by immunohistochemical(IHC)staining for 5-bromo-2'-deoxyuridine(BrdU)and Ki67 as well as hematoxylin and eosin(H&E)staining.Macrophage and neutrophil infiltration into livers were reflected by IHC staining for galectin-3 and myeloperoxidase(MPO)as well as flow cytometry analysis.Macrophage migration ability was evaluated by transwell assay.The mRNA levels for cell cycle or inflammation-related genes were measured by quantitative real-time RT-PCR(qPCR).The protein levels of cell proliferation related protein and phosphorylated signal transducer and activator of transcription 3(STAT3)were detected by Western blotting.Results:Ppara^(Mye−/−)mice showed enhanced hepatocyte proliferation and mitosis at 32 h after PHx compared with Ppara^(fl/fl)mice,which was consistent with increased proliferating cell nuclear antigen(Pcna)mRNA and cyclinD1(CYCD1)protein levels in Ppara^(Mye−/−)mice at 32 h after PHx,indicating an accelerated liver regeneration in Ppara^(Mye−/−)mice.IHC staining showed that macrophages and neutrophils were increased in Ppara^(Mye−/−)liver at 32 h after PHx.Livers of Ppara^(Mye−/−)mice also showed an enhanced infiltration of M1 macrophages at 32 h after PHx.In vitro,Ppara-deficient bone marrow-derived macrophages(BMDMs)exhibited markedly enhanced migratory capacity and upregulated M1 genes Il6 and Tnfa but downregulated M2 gene Arg1 expressions.Furthermore,the phosphorylation of STAT3,a key transcript factor mediating IL6-promoted hepatocyte survival and proliferation,was reinforced in the liver of Ppara^(Mye−/−)mice after PHx.Conclusions:This study provides evidence that myeloid PPARαdeficiency accelerates PHx-induced liver regeneration via macrophage polarization and consequent IL-6/STAT3 activation,thus providing a potential target for manipulating liver regeneration.

Severe acute respiratory syndrome coronavirus protein 6 mediates ubiquitin-dependent proteosomal degradation of N-Myc(and STAT) interactor

Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.

Study on the mechanism of Fuzi in the treatment of allergic rhinitis based on network pharmacology and experimental validation

Objective:To study the key target genes and signaling pathways in the treatment of Allergic Rhinitis(AR)with Radix Aconiti Lateralis Preparata(aka Fuzi).Methods:The TCMPS and PubChem databases were used to screen the active ingredients and target genes of Fuzi using oral bioavailability and drug similarity as screening conditions,and the GeneCards database was used to screen the target genes of AR.The online tool Venny2.1 was used to screen the target genes of Fuzi for the treatment of Allergic Rhinitis;the STRING database was used to obtain the protein-protein interaction(PPI)network of drug-disease targets,and the key target genes were identified by the MCC algorithm.The potential biological processes and signaling pathways were identified by GO enrichment and KEGG enrichment analysis.Finally,animal experiments were conducted to demonstrate the therapeutic effect ofFuzi on Allergic Rhinitis.Results:The TCMSP,PubChem and GeneCards databases were used to screen the 21 active compound components of Fuzi and 68 potential therapeutic target genes of Fuzi for Allergic Rhinitis.PPI network analysis identified the top ten key target genes,namely:PTGS2,TNF,IL6,AKT1,ALB,STAT3,CCL2,CXCL8,VEGFA and JUN,GO functional and KEGG pathway enrichment analysis showed that the significantly enriched functions and pathways of Fuzi on Allergic Rhinitis were closely related to Allergic Rhinitis.Finally,animal experiments were conducted to verify that Fuzi is effective in the treatment of Allergic rhinitis.Conclusion:Increased expression of IL-6 and TNF-αin nasal mucosal tissues of patients with Allergic Rhinitis was positively correlated with indicators related to the disease activity of AllergicRhinitis.Fuzi ameliorated the inflammatory changes in mice with Allergic Rhinitis by inhibiting the activation of Toll-like signaling pathway in the nasal mucosa and decreasing the expression activity of IL-6 and TNF-α.

A mussel-inspired,antibacterial,antioxidant,injectable composite hydrogel for the sustain delivery of salvianolic acid B for the treatment of frozen shoulder

Frozen shoulder(FS)manifests as progressively worsening pain and a reduction in shoulder range of motion(ROM).Salvianolic acid B(SaB)is recently expected to be used in the treatment of fibrosis diseases including FS.We firstly demonstrate that SaB can effectively hinder the progression of oxidative stress,inflammation,and pathological fibrosis within the synovial tissue in FS,potentially leading to the reduction or reversal of capsule fibrosis and joint stiffness.For further clinical application,we design and synthesize a novel,superior,antioxidant and antibacterial CSMA-PBA/OD-DA(CPDA)hydrogel for the delivery of SaB.In vitro experiments demonstrate that the CPDA hydrogel exhibits excellent biocompatibility and rheological properties,rendering it suitable for intra-articular injections.Upon injection into the contracted joint cavity of FS model rat,the SaBCPDA hydrogel accelerate the recovery of ROM and exhibit superior anti-fibrosis effect,presenting the promise for the treatment of FS in vivo.

Mechanisms of Shufeng Jiedu Capsule in treating bacterial pneumonia based on network pharmacology and experimental verification

Objective: The aim of this study was to investigate the underlying mechanism of Shufeng Jiedu Capsule(SFJD) for treating bacterial pneumonia(BP) in vivo based on network pharmacology and experimental verification study.Methods: Network pharmacology was used to screen the active compounds and target genes of SFJD.Then, the multi drug resistance-Pseudomonas aeruginosa(MDR-PA) mice lethal model and MDR-PA pneumonia model were established to evaluate the therapeutic effects and underlying mechanisms of SFJD.Western blot and ELISA were used to determinate the protein expression level of the IL-17 signaling pathway and JAK/STAT signaling pathway.Results: After screening, 172 potential components of SFJD were generated, based on which we constructed an SFJD-component-target-BP interaction network. The Gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) enrichment revealed that SFJD could regulate the IL-17 signaling pathway and Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway.Molecular docking showed that the potential target proteins had good combinations with the main active components. SFJD significantly reduced the mortality and prolonged survival days in lethal models. The lung index and pathological changes in the lung were also significantly decreased. SFJD could significantly decrease the expression of interleukin-17A(IL-17A), TNF receptor associated factor 6(TRAF6),phospho-inhibitor of nuclear factor-kappa B(p-IκB)/inhibitor of NF-κB(IκB), phospho-NF-κB p65(pNF-κB p65), phospho-protein kinase B(p-AKT)/AKT, phospho-signal transducer and activator of transcription 3(p-STAT3)/STAT3, phospho-signal transducer and activator of transcription 1(p-STAT1)/STAT1, and the protein level of interleukin-6(IL-6), tumor necrosis factor a(TNF-a), and IL-1β.Conclusion: Combined with network pharmacology and in vivo study, it was found that SFJD exerted its therapeutic effects on BP by inhibiting the IL-17 pathway and JAK/STAT signaling pathway. This study provides new evidence for SFJD in treatment of BP.