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Aim To investigate the effect of paeoniflorin(PF)on TLR2/4 pathway in AGEs-induced RAW264.7 macrophages.Methods RAW264.7 macrophages were incubated at different time points in AGEs stimulation,as well as different concentrations of PF,to optimize experimental conditions.RAW264.7 macrophages were randomly divided into five groups: control group(DMEM),bull serum albumin(BSA)group(200 mg·L-1 BSA),AGEs group(200 mg·L-1 AGEs),paeoniflorin group(200 mg·L-1 AGEs+10-5 mol·L-1 PF)and TLR2/4 inhibitor group(200 mg·L-1 AGEs+30 mg·L-1 OxPAPC).The expression of Toll-like receptor 2(TLR2),Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),p-IRAK1,TIR-domain containing adaptor protein-inducing IFN-β(TRIF),interferon regulatory factor 3(IRF3),p-IRF3,NF-κB p-p65,NF-κB p65,inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),interleukin-l β(IL-1β)and monocyte chemotactic protein-1(MCP-1)were measured by Western blot.Real-time PCR was used to detect the expression of TLR2 and TLR4 mRNA,while TNF-α,IL-1β and MCP-1 levels in cell supernatant were measured by ELISA.Results Compared with control group,AGEs significantly increased the expression of TLR2,TLR4,MyD88,p-IRAK1,TRIF,IRF3,p-IRF3,NF-κB p-p65,NF-κB p65,iNOS,TNF-α,IL-1β and MCP-1 proteins(P<0.01),as well as TLR2 and TLR4 mRNA(P<0.01).TNF-α,IL-1β and MCP-1 contents were also elevated in cell supernatant(P<0.01).The effects induced by AGEs were decreased significantly in PF and TLR2/4 inhibitor group(P<0.01).Conclusion PF plays an anti-inflammatory effect via inhibiting TLR2/4 pathway on macrophages,which may provide a new theoretical basis for the treatment of diabetic nephropathy.
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Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose.Methods Purity of mouse BMDM was detected by flow cytometry.The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol.Cells were divided into normal control group (RPMI 1640),osmolality control group (25 mmol/L mannitol),high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol).Immunocytochemistry and flow cytometry were used to detect macrophage subtype.The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR.Expressions of p-TAK1,TAK1 binding protein (TAB1),p-JNK,p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting.Results The purity of BMDM was about 99.36％.Compared with normal control group,high glucose group had increased percentage of M1 macrophages,increased expression of MCP-1 and TNF-α mRNA (all P ＜ 0.05).Moreover,p-TAK1,TAB1,p-JNK,p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P ＜ 0.05).After treatment with inhibitor 5Z-7-oxozeaenol,the effects induced by high glucose were inhibited (P ＜ 0.05).Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM,which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.
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Aim We used bone marrow-derived macro-phages ( BMMs ) , to explore the mechanism of macro-phage activation and the effect of TGF-β activated ki-nase-1 ( TAK1 ) inhibitor 5 Z-7-oxozeaenol on it under AGEs conditions. Methods The BMMs were obtained from C57 mice, and purity of BMMs was detected by flow cytometry. Cell viability was tested after treatment with different concentrations of TAK1 inhibitors. Laser confocal microscopy was used to detect macrophage M1 subtype . Flow cytometry was used to analyse the macro-phage activated by AGEs. TNF-α and MCP-1 mRNA levels were evaluated by qRT-PCR. Western blot was used to detect the expression levels of TAK1 signal pathway protein. Results AGEs stimulation could in-crese the activity of M1 macrophages,and 5Z-7-oxoze-aenol could inhibit the differentiation of BMMs. Com-pared with control group, AGEs increased the expres-sion of MCP-1 and TNF-α mRNA(P NF-κBp65 proteins ( P <0. 05 ) . Conclusions AGEs can induce BMMs to M1 phenotypic polarization. 5Z-7-oxozeaenol reduces the expression of inflammatory cyto-kine via inhibiting TAK1/MAPKs, MAPKs/NF-κB pathways.
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Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys.Methods The 48 10-week-old male db/db mice were randomly divided into db/db group,db/db+MT 50 μg/kg group,db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group,each consisting of 12 mice.These mice received i.p.injections of MT These mice received i.p.injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution,given every day].Alternatively,12 db/m mice served as the control group.db/m and db/db group were injected i.p.with the same volume of PBS/DMSO solution.The animals were sacrificed after 12 weeks of dosage administration.Blood glucose (BG),body weight (BW),kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined;Kidney pathological lesions were evaluated by renal pathological staining.Immunohistochemistry of renal TLR4,NF-κB p65,and ED-1 was performed to determine the immunoreactivity.Western blotting was used to detect the expression of renal TLR4,myeloid differentiation factor 88 (MyD88),TIR-domaincontaining adaptor inducing interferon-β (TRIF),interferon regulatory factor 3 (IRF-3) and NF-κB p65,while the mRNA expressions of renal tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR.Results Compared with control group,the levels of BG,BW,KW and UAER were much higher in db/db mice group (P ＜ 0.01),while KW in db/db+MT (100,200 μg/kg) groups and UAER level in db/db+MT (50,100,200 μg/kg) groups were distinctly decreased compared with those in db/db group (P ＜ 0.01).In week 12 db/db mice,the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P ＜ 0.01).The above kidney histopathologic lesions were distinctly ameliorated by 50,100,200 μg/kg MT (P ＜ 0.05).Immunohistochemistry intensity of renal TLR4,NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P ＜ 0.01),and that were remarkably decreased in db/db+MT (50,100,200 μg/kg) mice compared with db/db mice (P ＜ 0.05).Western blotting showed that the protein expression of renal TLR4,MyD88,TRIF,IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P ＜ 0.05) and weaker in db/db+ MT (50,100,200 μg/kg) groups compared with those in db/db group (P ＜ 0.05).Futhermore,the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P ＜ 0.01) and lower in db/db+MT (50,100,200 μg/kg) groups compared with those in db/db group (P ＜ 0.01).Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.
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Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism.Methods Twenty-four male db/db mice were randomly divided into two groups:db/db mice (db/db,n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ,n=12).Another group of wild type mice (n=12) was held as the control group.OZ 2 mg/kg was administrated by intraperitoneal injection every other day.At week 8 and 12 after 5Z-7-oxozeaenol treatment,blood glucose (BG),body weight (BW),kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated.Kidney pathological lesions were detected by light and electron microscopy.NF-κB p65,monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-ot (TNF-o) were detected by immunohistochemistry.Western blotting was used to detect p-TAK1,TAB1,p-p38MAPK and IL-1β expression,while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR.Results Compared with control group,the levels of BG,BW,KW and UAER were higher (P ＜ 0.01) in db/db mice group,while BW,KW and UAER levels were significantly decreased in db/db + OZ group compared with that in db/db mice group (P ＜ 0.05).In week 8 and 12 db/db mice,glomerular volume and extracellular matrix were increased,while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor.Immunohistochemistry showed that NF-κB p65,MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P ＜ 0.05) and were significantly inhibited by TAK1 inhibitor (P ＜ 0.05).Western blotting showed that p-TAK1,TAB1,p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P ＜ 0.05) and lower in db/db+ OZ group than that in db/db mice group (P ＜ 0.05).Moreover,real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P ＜0.05).Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.