Effect of Total Glucosides of Paeony on the Expression of Nephrin in the Kidneys from Diabetic Rats

2009 ◽  
Vol 37 (02) ◽  
pp. 295-307 ◽  
Author(s):  
Pei Zhang ◽  
Jing-Jing Zhang ◽  
Jing Su ◽  
Xiang-Ming Qi ◽  
Yong-Gui Wu ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transmembrane Protein ◽  
Chinese Herb ◽  
Diabetic Rats ◽  
Normal Group ◽  
Total Glucosides Of Paeony ◽  
Tnf Α

Total glucosides of paeony (TGP), extracted from the traditional Chinese herb root of Paeonia lactiflora pall, have been shown to have a therapeutic role in experimental diabetic nephropathy including albuminuria. Recent investigation has identified nephrin, a podocyte-specific transmembrane protein, as a key regulator in the pathogenesis of diabetic albuminuria. The aim of this study was to investigate whether TGP can attenuate albuminuria through prevention of nephrin loss in the experimental diabetic nephropathy. Fifty male Munich-Wistar rats were obtained from the Experimental Animal Center of Anhui Medical University. These rats were divided into 5 groups (n = 10); normal group, control diabetic group, and 3 TGP treated diabetic groups at different concentrations. Diabetes was induced by streptozotocin, and TGP (50, 100, 200 mg/kg) was orally administered to the 3 TGP treated diabetic groups once a day for 8 weeks, respectively. Blood glucose and 24 hour urinary albumin excretion rate (AER) were measured. The expressions of nephrin, tumor necrosis factor-α (TNF-α), NF-κB p65 and 3-nitrotyrosine (3-NT) protein were determined by immunoinfluorescence or Western blot analysis in the kidneys. Elevated AER was markedly attenuated by TGP treatment in diabetic rats. There was a finely dotted linear epithelial staining of nephrin in normal group glomeruli. In contrast, the staining of glomeruli from untreated diabetic rats was attenuated, more diapersed, and clustered. This diabetic-induced loss of glomerular nephrin expression was prevented in a large degree in TGP-treated diabetic rats. Western blot analysis showed that the expression of nephrin protein was reduced in the kidneys of diabetic rats, but significantly increased in the TGP treatment groups. The expressions of TNF-α, NF-κB p65 and 3-NT protein were significantly increased in the kidneys of diabetic rats, which were all significantly inhibited by TGP treatment. Our results showed that TGP could decrease AER in diabetic rat, and that its mechanism may be at least partly correlated with upregulation of the expression of nephrin in the kidney.

Total Glucosides of Paeony Regulates JAK2/STAT3 Activation and Macrophage Proliferation in Diabetic Rat Kidneys

2012 ◽  
Vol 40 (03) ◽  
pp. 521-536 ◽  
Author(s):  
Kun Wang ◽  
Yong-Gui Wu ◽  
Jing Su ◽  
Jing-Jing Zhang ◽  
Pei Zhang ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Albumin Excretion Rate ◽  
Janus Kinase ◽  
Diabetic Rat ◽  
Protective Effects ◽  
Active Constituent ◽  
Total Glucosides Of Paeony

Total glucosides of paeony (TGP) is the major active constituent of Paeonia lactiflora Pall., which has shown renoprotection in experimental diabetic nephropathy. Activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important mechanism by which hyperglycemia contributes to renal damage. Macrophages also play an essential role in the pathogenesis of diabetic nephropathy. Herein, we investigated the ability of TGP to modulate JAK2/STAT3 activation and macrophage proliferation in rats with streptozotocin (STZ)-induced diabetes. TGP (50, 100, and 200 mg/kg) was administered orally once a day for eight weeks. Levels of p-JAK2 and p-STAT3 were determined by Western blot analysis. Immunohistochemistry and double immunohistochemistry were used to identify p-STAT3, ED-1, PCNA/ED-1, and p-STAT3/ED-1-positive (+) cells. The elevated 24-h urinary albumin excretion rate was markedly attenuated by treatment with 50, 100, and 200 mg/kg TGP. Western blot analysis showed that the significantly increased levels of p-JAK2, p-STAT3 proteins in the kidneys of diabetic rats were significantly inhibited by 50, 100, and 200 mg/kg TGP treatment. The marked accumulation and proliferation of macrophages in diabetic kidneys were significantly inhibited by TGP treatment. ED-1+/p-STAT3+ cells were significantly increased in the kidneys from the model group but were significantly inhibited by TGP treatment. These results show that TGP significantly inhibited diabetic nephropathy progression and suggest that these protective effects are associated with the ability of TGP to inhibit the JAK2/STAT3 pathway and macrophage proliferation and action.

scholarly journals Poly(Adenosine 5′-Diphosphate-Ribose) Polymerase Inhibition Counteracts Multiple Manifestations of Experimental Type 1 Diabetic Nephropathy

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5273-5283 ◽  
Author(s):  
Viktor R. Drel ◽  
Weizheng Xu ◽  
Jie Zhang ◽  
Ivan A. Pavlov ◽  
Hanna Shevalye ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Diabetic Nephropathy ◽  
Western Blot ◽  
Blot Analysis ◽  
Renal Cortex ◽  
Parp Inhibitors ◽  
Diabetic Rats ◽  
Parp Inhibition ◽  
Tnf Α

Abstract This study was aimed at evaluating the role for poly(ADP-ribose) polymerase (PARP) in early nephropathy associated with type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with one of two structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427), at 3 mg/kg−1 · d−1 ip and 30 mg/kg−1 · d−1, respectively, for 10 wk after the first 2 wk without treatment. PARP activity in the renal cortex was assessed by immunohistochemistry and Western blot analysis of poly(ADP-ribosyl)ated proteins. Variables of diabetic nephropathy in urine and renal cortex were evaluated by ELISA, Western blot analysis, immunohistochemistry, and colorimetry. Urinary albumin excretion was increased about 4-fold in diabetic rats, and this increase was prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-associated increase in poly(ADP-ribose) immunoreactivities in renal glomeruli and tubuli and poly(ADP-ribosyl)ated protein level. Renal concentrations of TGF-β1, vascular endothelial growth factor, endothelin-1, TNF-α, monocyte chemoattractant protein-1, lipid peroxidation products, and nitrotyrosine were increased in diabetic rats, and all these changes as well as an increase in urinary TNF-α excretion were completely or partially prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-induced up-regulation of endothelin (B) receptor, podocyte loss, accumulation of collagen-α1 (IY), periodic acid-Schiff-positive substances, fibronectin, and advanced glycation end-products in the renal cortex. In conclusion, PARP activation is implicated in multiple changes characteristic for early nephropathy associated with type 1 diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

Adenosine monophosphate-activated protein kinase activator inhibits activation of fibroblast-like synoviocytes but promotes hyaluronan and proteoglycan link protein 1 secretion

2020 ◽  
Author(s):  
Yong Chen ◽  
Qiu Fujuan ◽  
Yu Beijia ◽  
Zuo Fangfang ◽  
Bi Yanan ◽  
...  
Keyword(s):  
Western Blot ◽  
Rna Sequencing ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Enrichment Analysis ◽  
Protective Mechanism ◽  
Sequencing Data ◽  
Positive Correlation ◽  
Tnf Α ◽  
Fibroblast Like Synoviocytes

Abstract Objectives: To determine whether any correlation exists between disease activity and AMPK levels in rheumatoid arthritis (RA) patients and investigate the effects of AMPK activator treatment on RA fibroblast-like synoviocytes (RA-FLS).Methods: Serum AMPK-α1, p-AMPK-α1, TNF-α and IL-17 levels between osteoarthritis (OA) and RA patients having different disease activities were compared by ELISA. Differentially expressed genes (DEGs) between RA and OA synovium from NCBI GEO Profiles (accession numbers: GSE1202112, GSE55235, GSE5545713) were identified and the genes intersecting in all the three datasets were selected for enrichment analysis. Immunohistochemical staining was done with synovium obtained from OA and RA patients for p-AMPK-α1. AMPK gene expression in synovium was semi-quantified by RT-qPCR. RNA sequencing of FLS was performed and DEGs were selected for KEGG enrichment analysis. AMPK activator, metformin, treated RA-FLS were tested for proliferation and migration by MTT and scratch test, respectively. Expression of IL-6, AMPK-α1, PKA-α, RAPTOR, mTOR, HAPLN1, RUNX1 and RUNX2 genes were determined by qPCR. Phosphorylated AMPK-α1 and HAPLN1 levels were determined by an automated electrophoresis-western blot analysis method.Results: In RA sera, a positive correlation between p-AMPK-α1 levels and DAS28 (r = 0.270, 95%CI: 0.142-0.492, p < 0.0001) as well as CRP levels (r = 0.259, 95%CI: 0.009-0.478, p < 0.05) was found. Similarly, a positive correlation was observed between AMPK-α1 and TNF-α levels (r = 0.460, 95% CI: 0.241-0.640, p = 0.0002). DEGs between OA and RA synovium from NCBI GEO profiles and our RNA sequencing data suggested activation of metabolic pathways specific to RA-FLS. AMPK-α1 was highly expressed in the synovium of RA but not OA patients. Metformin at higher concentrations inhibited RA-FLS proliferation in a dose dependant manner, however, at lower concentrations it has an opposite effect. On the other hand, AMPK inhibitor, dorsmophin, promoted the proliferation of RA-FLS significantly. Interestingly, both metformin and dorsmophin substantially inhibited the migration of RA-FLS. In FLS, relative expression level of IL-6 mRNA was significantly decreased after metformin treatment, while the expression of AMPK-α1, PKA-α and HAPLN1 genes were significantly increased. Western blot analysis confirmed increased expression of p-AMPK-α1 and HAPLN1 genes in the metformin treated FLS.Conclusions: Inflammatory stress in RA synovium leads to an increase in AMPK levels, possibly as a protective mechanism. AMPK activator but not metformin per se could be a potential therapeutic for RA by promoting HAPLN1 secretion to protect the joints.

scholarly journals Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro

2016 ◽  
Vol 19 (3) ◽  
pp. 485-494 ◽  
Author(s):  
R. Lin ◽  
Q. Wang ◽  
B. Qi ◽  
Y. Huang ◽  
G. Yang
Keyword(s):  
Immune Response ◽  
Western Blot ◽  
Alveolar Macrophages ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Splenic Lymphocytes ◽  
Rt Pcr ◽  
Tnf Α ◽  
Pulmonary Alveolar Macrophages

Abstract Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells

2019 ◽  
Vol 51 (10) ◽  
pp. 1056-1063 ◽  
Author(s):  
Jiaojiao Pu ◽  
Rui Wang ◽  
Guanglin Zhang ◽  
Ju Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hacat Cells ◽  
Tnf Α

Abstract The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin–eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

scholarly journals Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

2022 ◽  
Vol 12 ◽  
Author(s):  
Ahmed M. Abu El-Asrar ◽  
Ajmal Ahmad ◽  
Mohd Imtiaz Nawaz ◽  
Mohammad Mairaj Siddiquei ◽  
Alexandra De Zutter ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Matrix Metalloproteinase ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tissue Inhibitor ◽  
Matrix Metalloproteinase 3 ◽  
Tnf Α ◽  
Brb Breakdown

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion:In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

scholarly journals Clarithromycin Inhibits NF-κB Activation in Human Peripheral Blood Mononuclear Cells and Pulmonary Epithelial Cells

2001 ◽  
Vol 45 (1) ◽  
pp. 44-47 ◽  
Author(s):  
Takashi Ichiyama ◽  
Miki Nishikawa ◽  
Tomomi Yoshitomi ◽  
Shunji Hasegawa ◽  
Tomoyo Matsubara ◽  
...  
Keyword(s):  
Western Blot ◽  
Proinflammatory Cytokines ◽  
Peripheral Blood Mononuclear Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mononuclear Cells ◽  
A549 Cells ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Blood Mononuclear Cells

ABSTRACT Macrolide antibiotics modulate the production of proinflammatory cytokines in vivo and in vitro. Transcription of the genes for these proinflammatory cytokines is regulated by nuclear factor κB (NF-κB). We examined whether or not clarithromycin inhibits the activation of NF-κB induced by tumor necrosis factor alpha (TNF-α) or staphylococcal enterotoxin A (SEA) in human monocytic U-937 cells, a T-cell line (Jurkat), a pulmonary epithelial cell line (A549), and peripheral blood mononuclear cells (PBMC). Flow cytometry revealed that clarithromycin suppresses NF-κB activation induced by TNF-α in U-937 and Jurkat cells in a concentration-related manner. Western blot analysis also demonstrated that clarithromycin inhibits NF-κB activation induced by TNF-α in U-937, Jurkat, and A549 cells and PBMC and by SEA in PBMC. Western blot analysis of cytoplasmic extracts of A549 cells revealed that this inhibition is not linked to preservation of expression of the IκBα protein. The chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression is suppressed in U-937 cells pretreated with clarithromycin. These findings are consistent with the idea that clarithromycin suppresses the production of proinflammatory cytokines via inhibition of NF-κB activation.

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