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.

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.

Objective:To investigate the clinicopathological characteristics and influencing factors of kidney prognosis in primary IgA nephropathy (IgAN) patients.Methods:The data of primary IgAN patients diagnosed with renal biopsy in the First Affiliated Hospital of Anhui Medical University from January 2015 to September 2019 were retrospective analyzed. According to the level of baseline estimated glomerular filtration rate (eGFR) when performing renal biopsy, the patients were divided into group A[eGFR≥90 ml·min -1·(1.73 m 2) -1], group B[eGFR 61-89 ml·min -1·(1.73 m 2) -1] and group C[eGFR≤60 ml·min -1·(1.73 m 2) -1]. The clinical and pathological data were collected and compared among the three groups. Kaplan-Meier method was conducted for renal results, whereas the Cox proportional-hazards regression model was exploited to analyze the influencing factors of kidney prognosis in IgAN patients. Results:A total of 742 patients were included in the study, including 394 cases (53.1%) in group A, 203 cases (27.4%) in group B, and 145 cases (19.5%) in group C. There were 325 males (43.8%) and 417 females (56.2%). The median duration of renal biopsy was 6 (1, 24) months, and the median age was 36 years old (18-68 years old). As the baseline level of renal function decreased, the proportion of patients with nephrotic syndrome, hypertension, anemia and hyperuricemia and the levels of 24 h urinary protein, serum triglyceride and total cholesterol increased significantly (all P<0.05), while the proportion of gross hematuria episodes and the ratio of serum albumin to globulin significantly decreased (all P<0.05). For the aspect of pathological manifestations, the proportions of cell proliferation in capillaries (E1), segmental sclerosis or adhesion (S1), renal tubular atrophy or interstitial fibrosis (T1/2), globular sclerosis, renal arteriole wall thickening and vitreous degeneration, Lee's grade Ⅳ and Ⅴ increased with the decrease of baseline renal function (all P<0.05). Kaplan-Meier analysis showed that the cumulative renal survival rate decreased with the decline of baseline renal function (Log-rank χ2=88.510, P<0.001). As a result of multivariate Cox regression analysis, nephrotic syndrome ( HR=2.399, 95% CI 1.054-5.459, P=0.037), hypertension ( HR=1.806, 95% CI 1.071-3.048, P=0.027), low baseline eGFR (taking group A as the reference, group B: HR=2.383, 95% CI 1.053-5.392, P=0.037; group C: HR=6.878, 95% CI 3.074-15.393, P<0.001), IgG deposition ( HR=2.224, 95% CI 1.384-3.574, P=0.001) and globular sclerosis ( HR=2.075, 95% CI 1.230-3.501, P=0.006) were the independent influencing factors for renal progression in primary IgAN patients. Conclusions:The level of baseline renal function in primary IgAN patients can be used to predict the extent of clinic-pathological damage. Nephrotic syndrome, hypertension, low baseline eGFR, IgG deposition and globular sclerosis are the independent influencing factors for renal progression in primary IgAN patients.

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<0. 01). p-TAK1, TAB1,p-JNK,p-p38MAPK and NF-κBp65 proteins ex-pression also increased significantly ( P <0. 05 ) . After treatment with inhibitor, transcription levels of MCP-1 and TNF-α decreased significantly ( P < 0. 05 , P <0. 01 ) . 5 Z-7-oxozeaenol treatment downregulated the expression of p-TAK1,TAB1,p-JNK,p-p38MAPK and
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.

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.

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.

Purpose To investigate the clinical characteristics and Oxford classification of IgA nephropathy patients with hyperurice-mia. Methods 151 IgA nephropathy patients confirmed by renal biopsy in 2013 were analyzed retrospectively. The patients were di-vided into the two groups:IgA nephropathy patients with or without hyperuricemia. Morphological changes were evaluated with Oxford classification scoring system and Lee’s grades. A comparative analysis of clinical manifestations and pathological injuries was performed between the two groups. Results Incidence of hyperuricemia in IgA nephropathy patients was 48. 3% and was more common in young men. Hypertension was associated with hyperuricemia. Oxford classification of IgA nephropathy patients with hyperuricemia was pre-dominant M1E0S1T0 and Lee’s grades presented with grade Ⅲ. The outstanding histopathologic features with higher plasma uric acid levels indicated higher tubulointerstitial chronicity, higher glomerular sclerosis ratio, accompanied by a decline in glomerular filtration rate. There was no significant difference of vascular lesions. Conclusions The prevalence of hyperuricemia in IgA nephropathy pa-tients is high. Oxford classification shows IgA nephropathy with hyperuricemia are associated with more severe tubulointerstitial lesions and lower GFR.

BACKGROUND:In vitro studies have demonstrated that basic fibroblast growth factor (bFGF) promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells. However, it is unclear whether coronary venous retroperfusion of bFGF stimulates BMSCs differentiation in vivo. OBJECTIVE:To evaluate the effects of coronary venous retroperfusion of bFGF on BMSCs differentiation in vivo. METHODS:BMSCs from 12 dogs were isolated by density gradient centrifugation and expanded in vitro. These cells were transfected by enhanced green fluorescence protein (EGFP) lentiviral vector and the transfection efficiency was analyzed. Acute myocardial infarction was induced by ligation of left anterior descending coronary artery. After 1 week, 10 survival animals were randomized to BMSCs group (n=5) and bFGF+BMSCs group (n=5). bFGF-and EGFP-positive BMSCs were reversely infused via coronary vein using over-the-wire bal oon catheter. One week after infusion, the number of EGFP-positive cells co-staining factor VIII and troponin I was compared between the two groups by immunofluorescence method. RESULTS AND CONCLUSION:BMSCs were successful y transfected by EGFP and the transfection efficiency was 85%. Immunofluorescence showed that EGFP-positive BMSCs were observed in 23.5%of slides. There were more EGFP-positive cells co-staining VIII and troponin I in the bFGF+BMSCs group than in the BMSCs group (P<0.05). Thus, the coronary venous retroperfusion of bFGF enhances the differentiation of BMSCs into vascular endothelial cells and cardiomyocytes. Combined delivery of bFGF and BMSCs can exert a synergistic effect to promote cardiac repair.

10.3969/j.issn.2095-4344.2013.24.015

Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells.Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes.Exosomes were identified by transmission electron microscope and Western blotting.(2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage.Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes.The expression of inducible nitric oxide synthase (iNOS),tumor necrosis factor-α (TNF-α),Interleukin-1β (IL-1β),monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA),and the expression of p-c-Jun NH2-terminal kinase (p-JNK),mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot.Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63,TSG101 but absence of calnexin which is a marker of endoplasmic reticulum,suggesting that the exosomes were not contaminated with cells.(2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages.Compared with normal glucose exosomes group,high glucose exosomes had increased the expression of iNOS,TNF-α,IL-1β and MCP-1 in THP-1 macrophages (all P ＜ 0.01),moreover,p-JNK,p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P ＜ 0.01).Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.