Exploring the effect of Bushen Bitong recipe-containing serum on IL-1β-induced chondrocyte apoptosis based on SOX9/NF-κB/MMP-13 signaling pathway

Objective:To observe the effect and possible mechanism of action of Bushen Bitong recipe(BSBT)containing serum on IL-1β-induced chondrocyte apoptosis.Methods:Generation 3 rat chondrocytes were randomized into Control,IL-1β,IL-1β+BSBT(L),IL-1β+BSBT(M),and IL-1β+BSBT(H)groups(5%,10%and 15%BSBT-containing serum),and then 24h after intervention respectively,the cell proliferation and Apoptosis rate;Western blot detected the expression levels of Bcl-2,BAX,Caspase-3,SOX9,NF-κB p65,MMP-13 proteins in chondrocytes.ELISA detected the levels of TNF-α,IL-6,and bFGF in the supernatants of chondrocyte culture.Results:Compared with Control group,cell proliferation activity decreased,apoptosis rate increased,NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level increased,and SOX9 protein level and bFGF level decreased in IL-1βgroup;compared with IL-1βgroup,different concentrations of BSBT-containing serum group,cell proliferation activity increased,and apoptosis rate decreased.NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level decreased,SOX9 protein level and bFGF level increased;compared with IL-1β+BSBT(L)group,cell proliferation activity increased,apoptosis rate decreased in IL-1β+BSBT(M)and IL-1β+BSBT(H)groups,and NF-κB p65,MMP-13 protein level and TNF-αlevel decreased.13 protein levels and TNF-αand IL-6 levels decreased,and SOX9 protein levels and bFGF levels increased.Conclusion:BSBT-containing serum may promote IL-1β-induced proliferation of chondrocytes,reduce apoptosis,improve the microenvironment of chondrocytes,and promote cartilage repair through the SOX9/NF-κB/MMP-13 signaling pathway.

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.

The involvement of IL-23/Th17 pathway in mice virus myocarditis inducedby cosakievirus B3

Background The IL-23/ Th17 pathway plays an important role in the development of chronic in? ammatory diseases and autoimmune diseases.However,the role of the IL-23/Th17 axis in the regulation of virus myocarditis is still largely unknown.We aim to determine the role of IL-23/ Th17 axis in virus myocartidis.Methods and Results Balb/c male mice were peritoneally injected with 100TICD50 Coxsackie virus B3 to establish virus myocarditis(VMC), mice injected with PBS peritoneally were taken as the controls. 0,1,2,3,4 and 6 weeks After injection,IL-23,L-17 and RORγt mRNA in the myocardium of VMC were assessed by Semi-quantitative RT-PCR,and IL-23 protein from plasm was evaluated by ELISA.Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in CD4,To investigated whether the IL-23 is important during IL-23/Th-17 pathway challenge,we isolated CD4 + T cells and cultured with rIL-23 in vitro,and examined the Th17 cells.Results show that,comparing with the controls,IL-23,IL-17 and RORγt mRNA was steadly expressed in the myocardiums of infected mice from 1 week after virus injection.Conclusions IL-23/ Th-17 pathway may therefore play an essential role in VMC. Comparative studies are required to reveal further the roles of these cytokines in the pathogenesis of? these immune-related diseases.

Ragweed pollen induces allergic conjunctivitis immune tolerance in mice via regulation of the NF-κB signal pathway

AIM:To investigate the feasibility and mechanism of immune tolerance in allergic conjunctivitis.METHODS:The allergic conjunctivitis immune tolerance mice model was established by ragweed pollen(RW)and the related cytokines were detected.The mice were divided into 9 groups and the maslinic acid(MA)or PBS were given for different group after modeling.The expression levels of chemokine ligand 5(CCL5)and P-65 in the conjunctival tissue were analyzed by immunohistochemistry,quantitative reverse transcription polymerase chain reaction(q RT-PCR)and Western blot.The percentage of interleukin-17(IL-17)and CD4+CD25+in the splenocyte supernatant was analyzed by flow cytometry.Fur thermore,the serum and splenocyte supernatant concentration of total-IgE,interleukin-10(IL-10),and IL-17 was analyzed by enzyme linked immune response(ELISA).RESULTS:After the model was established,symptoms of conjunctivitis were alleviated,the level of P-65,CCL5,IL-17,and total-IgE was raised,while the expression of IL-10,CD4+CD25+was decreased.This result fully demonstrated that a typical IL-17/regulatory-T-cells(Treg cells)imbalance and NF-κB activation.When the NF-κB signal pathway was suppressed,it showed that there was a further relief of conjunctivitis in mice.At the same time,the expression of total-IgE,IL-17,and CCL5 was decreased and the expression of anti-inflammatory factor(IL-10,CD4+CD25+)was increased.CONCLUSION:In the state of immune tolerance,symptoms of conjunctivitis in mice are alleviated,the Th-17 cells of allergic conjunctivitis mice are inhibited,and Treg cells activity is enhanced.

FXYD3 enhances IL-17A signaling to promote psoriasis by competitively binding TRAF3 in keratinocytes

Psoriasis is a common chronic inflammatory skin disease characterized by inflammatory cell infiltration and epidermal hyperplasia.However,the regulatory complexity of cytokine and cellular networks still needs to be investigated.Here,we show that the expression of FXYD3,a member of the FXYD domain-containing regulators of Na+/K+ATPases family,is significantly increased in the lesional skin of psoriasis patients and mice with imiquimod(IMQ)-induced psoriasis.IL-17A,a cytokine important for the development of psoriatic lesions,contributes to FXYD3 expression in human primary keratinocytes.FXYD3 deletion in keratinocytes attenuated the psoriasis-like phenotype and inflammation in an IMQ-induced psoriasis model.Importantly,FXYD3 promotes the formation of the IL-17R-ACT1 complex by competing with IL-17R for binding to TRAF3 and then enhances IL-17A signaling in keratinocytes.This promotes the activation of the NF-κB and MAPK signaling pathways and leads to the expression of proinflammatory factors.Our results clarify the mechanism by which FXYD3 serves as a mediator of IL-17A signaling in keratinocytes to form a positive regulatory loop to promote psoriasis exacerbation.Targeting FXYD3 may serve as a potential therapeutic approach in the treatment of psoriasis.

Epigenetic regulation of Wnt signaling pathway gene SRY-related HMG-box 17 in papillary thyroid carcinoma

Background SRY-related HMG-box 17 （SOX17） encodes a member of the SOX （SRY-related HMG-box） family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate. Recently, it was considered as a tumor suppressor gene to inhibit canonical Wnt/β-catenin signaling pathway in several malignancies. However, the function of SOX17 in thyroid cancer was unknown. Therefore, we investigated the epigenetic changes and the function of SOX17 in thyroid cancer. Methods The methylation status of the promoter region of SOX17 was detected using methylation-specific PCR in 63 papillary thyroid carcinoma （PTC） tissue, 10 normal thyroid tissue, and two thyroid cancer cell lines. Semi-quantitative RT-PCR was used to assess mRNA expression of SOX17 before and after 5-aza-2＇-deoxycytidine treatment in thyroid cancer cell lines. Expression of SOX17 and β-catenin were detected by immunohistochemistry in PTC and adjacent tissue. Luciferase reporter assay, colony formation, transfection, and Western blotting were employed to analyze the effect of SOX17 on thyroid cancer cell proliferation and the function of SOX17 in the Wnt signal pathway. Results Loss of SOX17 expression was correlated to the promoter region hypermethylation in thyroid cancer cell lines. Re-expression of SOX17 was found in TPC-1 cell line after 5-aza-2＇-deoxycytidine treatment. In primary thyroid cancer, 60.3% （38/63） were methylated and 39.7% （25/63） unmethylated. But no methylation was found in noncancerous thyroid tissues. Methylation of SOX17 was associated reversely with β-catenin expression in the cytoplasm or nucleus significantly in the PTC （P 〈0.05）. Colony formation was inhibited by re-expression of SOX17 in TPC-1 cells. SOX17 suppressed the Wnt signaling pathway and the HMG domain was essential for this effect. Conclusions SOX17 was frequently methylated in human PTC. Loss of SOX17 expression was induced by promoter region hypermethylation. SOX17 inhibited thyroid cancer proliferation. Methylation of SOX17 activated the Wnt signaling pathway in human thyroid cancer.

The IL-1R/TLR signaling pathway is essential for efficient CD8+ T-cell responses against hepatitis B virus in the hydrodynamic injection mouse model

The outcome of hepatitis B viral(HBV)infection is determined by the complex interactions between replicating HBV and the immune system.While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively,the contribution of innate immune mechanisms remains to be defined.Here we examined the role of the interleukin-1 receptor/Toll-like receptor(IL-1R/TLR)signaling pathway in adaptive immune responses and viral clearance by exploring the HBV mouse model.Hydrodynamic injection with a replication-competent HBV genome was performed in wild-type mice(WT)and a panel of mouse strains lacking specific innate immunity component expression.We found higher levels of HBV protein production and replication in Tlr2^(−/−),Tlr23479^(−/−),3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice,which was associated with reduced HBV-specific CD8+T-cell responses in these mice.Importantly,HBV clearance was delayed for more than 2 weeks in 3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice compared to WT mice.HBV-specific CD8+T-cell responses were functionally impaired for producing the cytokines IFN-γ,TNF-αand IL-2 in TLR signaling-deficient mice compared to WT mice.In conclusion,the IL-1R/TLR signaling pathway might contribute to controlling HBV infection by augmenting HBV-specific CD8+T-cell responses.

Mechanisms of Up-regulation of 17β-Estrodiol on β-Defensin-2 ( SBD-2) Expression in Epithelial Cells of Ovine Oviduct

[ Objective] To investigate the mechanisms involved in the Up-regulatory effects of 17β-estrodiol on β-defensin-2 （SBD-2） in epithelial cells of ovine oviduct. [ Methods] Epithelial cells of ovine oviduct were isolated and cultured; and then the cultured cells at secondary generation were divided into 17β-estradiol （E2, 10^-8 tool/L） group, estrogen nuclear receptor antagonist ICI182780 （10^-7 tool/L） group, PKA antagonist KT-5720 （1 μmol/L） group, PKC antagonist H- 7（50 μmol/L） group, nuclear factor kappa B antagonist PDTC（50μmol/L） group and the blank control group （ Control ）. Firstly, different antagonists were added into corresponding antagonist groups in order to interfere the epithelial ceils of ovine oviduct for 1 h. Then, 17β-estradiol （ 10^-8 mol/L） was added into each antagonist group and E2 group for cultivation for 6 h. Finally, real-time fluorescent quantitative RT-PCR was used to detect the changes of SBD-2 mRNA expression. [ Results] 10^-8 mol/L 17β-estrodiol had significantly Up-regulatory effects on the expression of SBD-2 mRNA （P 〈 0. 05 ）. Estrogen nuclear receptor antagonist ICI182780, NF-κB antagonist PDTC and PKC antagonist H-7 could all block the Up-regnlatory effects on SBD-2. But PKA antagonist KT-5720 showed no significant effects on the Up-regulation of SBD-2 mRNA expression induced by 17β-estrodiol. [ Conclusions] SBD-2 mRNA expression induced by 17β-estrodiol in epithelial cells of ovine oviduct was mediated by estrogen nuclear receptor ICI182780, NF-κB and PKC pathways. However, PKA pathway might not participate in the Up-regulation of SBD-2 mRNA expression.

Mechanism of Liancao-Xieli capsule in the treatment of ulcerative colitis through the differentiation of Th17 and Treg cells

Objective:To observe the effect of Liancao-Xieli Capsule on STAT signal pathway and T cell differentiation in mouse model of ulcerative colitis;Methods:Forty BALB/c mice were randomly divided into the control group,model group,mesalazine group and Liancao-Xieli capsule group.Except the control group,the other three groups were treated with 3%dextran sodium sulfate free drinking water to construct the model of ulcerative colitis.During the modeling period,each group was given corresponding drugs for intervention,while the control group and the model group were given the same volume of distilled water by gavage as the control.After treatment,HE staining was used to observe the pathological changes of colon tissue,flow cytometry was used to detect the proportion of Th17 and Treg cells in spleen and mesenteric lymph nodes,and ELISA was used to detect TGF-β,IL-6 and IL-17A in colon tissue.Western blot was used to detect the expression levels of related proteins in STAT3/ROR-γt and STAT5/Foxp3 signaling pathways.Results:Compared with the model group,the body weight,colon length and the content of TGF-βin the colon tissue of the mice in the Liancao-Xieli capsule group increased significantly after the experiment,while the DAI score,colon histopathology score,and the contents of IL-6 and IL-17A in the colon tissue were significantly reduced,and the difference was statistically significant(P<0.01).At the same time,Liancao-Xieli capsule can reduce the ratio of Th17 cells and the ratio of Th17/Treg in the spleen and mesenteric lymph node tissues of UC mice,and increase the ratio of Treg cells,and the difference is statistically significant when compared with the model group(P<0.01).In addition,compared with the model group,the expression levels of p-STAT3 and RORγt protein in the colon tissue of the Liancao-Xieli capsule group were significantly reduced,and the expression levels of p-STAT5 and Foxp3 protein were significantly increased after treatment,and the differences are statistically significant(P<0.01),while the expression levels of STAT3 and STAT5 proteins in colon tissue did not change significantly,and the difference was not statistically significant(P>0.05);Conclusion:Liancao-Xieli Capsule can regulate the immune balance of Treg/Th17 and improve the intestinal inflammation of UC.Its mechanism of action is mainly through inhibiting STAT3/ROR-γt and promoting the activation of STAT5/Foxp3 signaling pathway.

Apoptotic Signal Pathways and Regulatory Mechanisms of Cancer Cells Induced by IL-24

The melanoma differentiation-associated gene-7（mda-7）,IL-24,has the specific functions that induce cancer cell apoptosis without doing harm to normal cells. We systematically review the apoptotic signal pathways and their regulatory mechanisms induced by Ad.IL-24 and IL-24 in diverse cancer cells. IL-24 can participate in varied signal transduction pathways,including JAK,p38 MAPK,Wnt/β-catenin,JNK,ER stress and mitochondria-associated signal pathways. And we review five proteins interacting with IL-24,including Bip/GRP78,S1 R,PKR,Beclin1 and soluble clusterin,which are relative to the tumor-specific effect of IL-24. It is speculated that ER stress,G-protein pathways and MAPK signal pathways may be the primary upstream effectors which activate the sequential downstream mediators resulting in apoptosis induced by IL-24 in tumor cells. Experimental results also show that IL-24 sensitizes cancer cells and indirectly promotes apoptosis rather than functions as a direct apoptosis inducer itself.

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.

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.

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.

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the func-tions of membrane receptors in T cells and suppressed T cell -mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n?3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study con-cluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.

Fhe roles and functional mechanisms of interleukin-17 amily cytokines in mucosal immunity

The mucosal immune system serves as our front-line defense against pathogens. It also tightly maintains immune tolerance to self-symbiotic bacteria, which are usually called commensals. Sensing both types of microorganisms is modulated by signalling primarily through various pattern-recognition receptors （PRRs） on barrier epithelial cells or immune cells. After sensing, proinflammatory molecules such as cytokines are released by these cells to mediate either defensive or tolerant responses. The interleukin-17 （IL-17） family members belong to a newly characterized cytokine subset that is critical for the maintenance of mucosal homeostasis. In this review, we will summarize recent progress on the diverse functions and signals of this family of cytokines at different mucosal edges.

A shedding soluble form of interleukin-17 receptor D exacerbates collagen-induced arthritis through facilitating TNF-α-dependent receptor clustering

Rheumatoid arthritis(RA)is exacerbated by TNF-alpha signaling.However,it remains unclear whether TNF-α-activated TNFR1 and TNFR2 are regulated by extracellular factors.Here,we showed that soluble glycosylated interleukin-17 receptor D(sIL-17RD),which was produced by proteolytic cleavage,enhanced TNF-α-induced RA.We revealed that IL-17RD shedding was induced by the proteolytic enzyme TACE and enhanced by TNF-αexpression in macrophages.Intriguingly,sIL-17RD was elevated in the sera of arthritic mice and rats.Recombinant sIL-17RD significantly enhanced the TNF-α-induced proinflammatory response by promoting TNF-α-TNFR-sIL-17RD complex formation and receptor clustering,leading to the accelerated development of collagen-induced arthritis.Our observations revealed that ectodomain shedding of IL-17RD occurred in RA to boost the TNF-α-induced inflammatory response.Targeting sIL-17RD may provide a new strategy for the therapy of RA.

A de novo missense mutation in MPP2 confers an increased risk of Vogt–Koyanagi–Harada disease as shown by trio-based whole-exome sequencing

Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

Contribution and underlying mechanisms of CXCR4 overexpression in patients with systemic lupus erythematosus

Aberrant expression of CXCR4 has been indicated to play a role in the pathogenesis of systemic lupus erythematosus(SLE),but the mechanism of CXCR4 dysregulation in SLE is unclear.This study is aimed to explore the clinical significance and possible mechanisms of abnormal CXCR4 expression on B cells from patients with untreated SLE.Expression of CXCR4 on peripheral B cells was determined by flow cytometry and western blotting.Freshly isolated B cells were cultured with exogenous interleukin 21(IL-21)in the presence or absence of CD40 ligand(CD40L)plus anti-IgM antibody(aIgM),and changes in CXCR4 expression were detected.Involvement of phosphatidylinositol 3 kinase(PI3K)/Akt and Janus kinase/Signal transducer and activator of transcription(JAK/STAT)signaling pathways was assessed by adding blocking agents Ly294002 and AG490.Since CD63 is reported to mediate endosomal recruitment of CXCR4 and BCL6 is capable of silencing CD63 gene transcription,we also measured BCL6 and CD63 gene transcription with real-time PCR.It was shown that CXCR4 expression on B cells was significantly upregulated in SLE patients,especially in those with lupus nephritis,and was positively correlated with SLE Disease Activity Index scores and negatively with the serum complement 3 levels(Po0.05).Downregulation of CXCR4 by IL-21 was intact.In contrast,a similar effect of aIgM plus CD40L in downregulating CXCR4 expression was defective in SLE patients but was restored by co-stimulation with IL-21 in vitro.Both Ly294002 and AG490 promoted downregulation of surface CXCR4 expression on B cells from SLE patients(P=0.078 and P=0.064).Furthermore,B cells from SLE patients exhibited diminished CD63 mRNA and enhanced BCL6 mRNA expression(both Po0.05).To sum up,CXCR4 was overexpressed on SLE B cells,positively correlating with disease activity and kidney involvement.Overactivation of the PI3K/Akt and JAK/STAT pathways as well as defective CD63 synthesis may contribute to CXCR4 dysregulation in SLE.

Transcriptome sequencing and metabolome analysis reveal the mechanism of Shuanghua Baihe Tablet in the treatment of oral mucositis

Oral mucositis(OM) caused by cancer therapy is the most common adverse reaction in the radiotherapy of head and neck tumors. In severe cases, it can lead to the interruption of treatment, which affects the control of the disease and the quality of life.Shuanghua Baihe Tablet(SBT) is a traditional Chinese medicine(TCM) formula, which is administerd to treat OM in China. It has been clinically effective for more than 30 years, but the underlying mechanism is not completely understood. With the development of multiple omics, it is possible to explore the mechanism of Chinese herbal compound prescriptions. Based on transcriptomics and metabolomics, we explored the underlying mechanism of SBT in the treatment of OM. An OM model of rats was established by 5-FU induction, and SBT was orally administered at dosages of 0.75 and 3 g·kg;·d;. In order to search for SBT targets and related metabolites, the dysregulated genes and metabolites were detected by transcriptomics and metabolomics. Immune related indicators such as interleukin-17(IL-17) and tumor necrosis factor-α(TNF-α) were detected by ELISA. Treg cell disorders was analyzed by flow cytometry. Our results showed that SBT significantly alleviated the symptoms of OM rats and the inflammatory infiltration of ulcer tissues.After SBT administration, inflammatory related metabolic pathways including linoleic acid metabolism, valine, leucine and isoleucine biosynthesis were significantly altered. Furthermore, the production of proinflammatory factors like IL-17 and TNF-α, were also dramatically reduced after SBT administration. Besides, the infiltration degree of Treg cells in the spleen of OM modeling rats was significantly improved by SBT administration, thus maintaining the immune balance of the body. The current study demonstrates that SBT regulates inoleic acid metabolism, glycerophospholipid metabolism and amino acid metabolism, and inhibits IL-17/TNF signal transduction to restore Treg and Th17 cell homeostasis in OM rats, thereby alleviating chemotherapy-induced OM.

A novel PI3K inhibitor XH30 suppresses orthotopic glioblastoma and brain metastasis in mice models

Glioblastoma is carcinogenesis of glial cells in central nervous system and has the highest incidence among primary brain tumors.Brain metastasis,such as breast cancer and lung cancer,also leads to high mortality.The available medicines are limited due to blood-brain barrier.Abnormal activation of phosphatidylinositol 3-kinases(PI3 K)signaling pathway is prevalent in glioblastoma and metastatic tumors.Here,we characterized a 2-amino-4-methylquinazoline derivative XH30 as a potent PI3 K inhibitor with excellent anti-tumor activity against human glioblastoma.XH30 significantly repressed the proliferation of various brain cancer cells and decreased the phosphorylation of key proteins of PI3 K signaling pathway,induced cell cycle arrest in G1 phase as well.Additionally,XH30 inhibited the migration of glioma cells and blocked the activation of PI3 K pathway by interleukin-17 A(IL-17 A),which increased the migration of U87 MG.Oral administration of XH30 significantly suppressed the tumor growth in both subcutaneous and orthotopic tumor models.XH30 also repressed tumor growth in brain metastasis models of lung cancers.Moreover,XH30 reduced IL-17 A and its receptor IL-17 RA in vivo.These results indicate that XH30 might be a potential therapeutic drug candidate for glioblastoma migration and brain metastasis.