| BackgroundDiabetic nephropathy (DN) is one of the most common chronic capillaries complications in patients with Diabetes mellitus (DM), its incidence increases with the disease course of DM lengthening, most patients of which are further developed to end-stage renal disease (ESRD) and chronic renal failure or even death.Though the precise pathogenesis of DN have not been clarified, several studies have already unraveled that glomerular hyperfiltration and mesangial expansion are the two characteristic pathophysiological changes in the early stage of DN. It is believed to be influenced by multiple determints with the high glucose level as the initiation factor. Strong oxidative stress is observed in DN and plays an important role in occurance and development of DN. The remarkable increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. NADPH oxidase is an important source of ROS production in MCs. Nox4is the main subunit of NADPH oxidase in MCs. Akt/NF-κB signaling pathway is also play an important role in DN and is associated with NADPH oxidase-derived ROS closely. TRPC6exists throughout the glomerulus in kidney tissues, especially in MCs. Hyperglycemia could down-regulate the expression of TRPC6protein, which resulted in the decrease of intracellular calcium, leading to the impaired MCs contractile function. Despite the research of drugs in prevention and treatment of DN is improving, it still calls for further development.Astragaloside Ⅳ (AS-Ⅳ), a purified small molecular saponin, is one of the main active ingredients of Radix Astragali, which has been reported to possess comprehensive biological properties, including antioxidant, anti-inflammation, immunoregulation, anti-aging and improving intellectual development. Recent studies have shown that AS-IV could ameliorate oxidative stress and proteinuria in STZ-induced diabetic rats. However, the protective effect and its precise mechanism of AS-IV on oxidative stress injury of MCs under high glucose conditions are not clearly understood. In this study, we aimed to examine whether high glucose activated NADPH oxidase/ROS/Akt/NF-κB pathway contribute to MCs proliferation and downexpression of TRPC6, and the effect of AS-Ⅳ on HG action in MCs.ObjectivesTo investigate the effects of AS-IV on HG-induced MCs proliferation and downregulation of TRPC6through a mechanism associated with the inhibition of NADPH oxidase-mediated ROS production, Akt, and NF-κB activation, and then to provide a novel therapeutic approach for the treatment of DN.Methods1. The cultured MCs were divided into3groups as follows:(1) normal control (NG:5.6mM glucose) group,(2) mannitol osmotic control (MA:NG+24.5mM mannitol) group,(3) high glucose (HG:25mM glucouse) group, after incubated for24h,48h,72h,96h, MTT assay was adopted to examine the OD value of each group.2. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG6h group,(3) HG12h group,(4) HG24h group,(5) HG36h group,(6) HG48h group. Real-time PCR method was used to detect the expression level of Nox4mRNA and TRPC6mRNA, and Western blot method was adopted to detect the expression of Nox4protein and TRPC6protein of each group. 3. The cultured MCs were divided into7groups as follows (1) NG group,(2) HG0.5h group,(3) HG1h group,(4) HG3h group,(5) HG12h group,(6) HG24h group,(7) HG48h group. Western blot method was used to detect the phosphorylation and degradation levels of IκBα of each group.4. The cultured MCs were divided into6groups as follows:(1) NG group,(2) NG+Tempol100μM group,(3) NG+DPI10μM group,(4) HG group,(5) HG+Tempol100μM group,(6) HG+DPI10μM group. After incubated for48h, using MTT assay to examine the OD value, using the fluorescent probe DCFH-DA to detect the intracellular ROS level, and using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity of each group.5. The cultured MCs were divided into6groups as follows:(1) NG group,(2) NG+FAM siRNA group,(3) NG+Nox4siRNA group,(4) HG group,(5) HG+FAM siRNA group,(6) HG+Nox4siRNA group. After incubated for48h, using MTT assay to examine the OD value of each group, using the fluorescent probe DCFH-DA to detect the intracellular ROS levels in each group, using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity, and using Western blot method to detect the expression levels of Nox4protein and TRPC6protein of each group.6. The cultured MCs were divided into6groups as follows:(1) NG group,(2) NG+FAM siRNA group,(3) NG+IκBα siRNA group,(4) HG group,(5) HG+FAM siRNA group,(6) HG+IκBα siRNA group. After stimulated for48h, using MTT assay to examine the OD value of each group, using the fluorescent probe DCFH-DA to detect the intracellular ROS level in each group, using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity, and using Western blot to detect the expression levels of Nox4protein and TRPC6protein, the phosphorylation levels of Akt and IκBα and the degradation level of IλBα protein in each group. 7. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+LY29400210μM group,(4) NG+DPI10μM group,(5) HG+Tempol100μM group,(6) HG+Sul500μM group, After stimulated for48h, using MTT assay to examine the OD value of each group, using the fluorescent probe DCFH-DA to detect the intracellular ROS level in each group, using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity, using Western blot method to detect the expression of Nox4protein and TRPC6protein, the phosphorylation levels of Akt and IκBα and the degradation level of IκBα protein in each group, and using Fluo-3/AM fluorescent probe to detect the free calcium concentration in each group.8. The cultured MCs were divided into9groups as follows:(1) NG group,(2) MA group,(3) HG group,(4) HG+AS-Ⅳ5μM group (5) HG+AS-Ⅳ10μM group,(6) HG+AS-Ⅳ25μM group,(7) HG+AS-Ⅳ50μM group,(8) HG+AS-Ⅳ100μM group,(9) HG+0.5%DMSO group. After incubated for48h, MTT assay was adopted to examine the OD value of each group.9. The cultured MCs were divided into9groups as follows:(1) NG group,(2) MA group,(3) HG group,(4) HG+AS-Ⅳ5μM group,(5) HG+AS-Ⅳ10μM group,(6) HG+AS-Ⅳ25μM group,(7) HG+AS-Ⅳ50μM group,(8) HG+AS-Ⅳ100μM group,(9) HG+0.5%DMSO group. After incubated for48h, calculating the ratio of total protein to cell number of each group.10. The cultured MCs were divided into8groups as follows:(1) NG group,(2) NG+AS-Ⅳ5μM group,(3) NG+AS-Ⅳ10μM group,(4) NG+AS-Ⅳ25μM group,(5) NG+AS-Ⅳ50μM group,(6) NG+AS-Ⅳ100μM group,(7) NG+0.5%DMSO group. After incubated for48h, MTT assay was adopted to examine the OD value of each group.11. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+AS-Ⅳ25μM group,(4) HG+AS-Ⅳ50μM group,(5) HG+AS- Ⅳ100μM group,(6) HG+Tempol100μM group, After incubated for48h, the fluorescent probe DCFH-DA was used to detect the intracellular ROS level of each group.12. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+AS-IV25μM group,(4) HG+AS-IV50μM group,(5) HG+AS-Ⅳ100μM group,(6) HG+OAG100μM group, After incubated for48h, using real-time PCR method to detect the expression level of TRPC6mRNA, using Western blot method to detect the expression level of TRPC6protein, and using Fluo-3/AM fluorescent probe to detect the free calcium concentration in each group.13. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+AS-Ⅳ25μM group,(4) HG+AS-Ⅳ50μM group,(5) HG+AS-Ⅳ100μM group,(6) HG+DPI10μM group, After incubated for48h, the cell NADPH oxidase colorimetric assay kit was usd to examine the NADPH oxidase activity, Western blot method was used to detect the expression level of Nox4protein, real-time PCR was used to detect the expression level of Nox4mRNA.14. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+AS-Ⅳ25μM group,(4) HG+AS-Ⅳ50μM group,(5) HG+AS-Ⅳ100μM group,(6) HG+LY2940010μM group, After incubated for48h, Western blot was adopted to detect the expression levels of Akt and p-Akt.15. The cultured MCs were divided into6groups as follows:(1) NG group,(2) HG group,(3) HG+AS-IV25μM group,(4) HG+AS-Ⅳ50μM group,(5) HG+AS-Ⅳ100μM group,(6) HG+Sul500μM group, After incubated for48h, Western blot was used to detect the expression levels of IκBα and p-IκBα.Results1. HG significantly stimulated MCs proliferation and hypertrophy in comparison to NG condition. Unlike HG, the addition of mannitol (MA) to the media did not display obvious influence on the proliferation and hypertrophy of MCs as compared with control, suggesting that the HG triggered MCs proliferation and hypertrophy are not the results of high osmolality within the media. MCs in HG condition for48h resulted in a significant increase in NADPH activtion and in ROS generation compared with NG. The HG action in MCs was notably abolished by the treatment of NADPH oxidase inhibitor (DPI,10μM) or ROS inhibitor (Tempol,100mM).2. The expression levels of Nox4protein and Nox4mRNA were upregulated significantly in HG condition. After Nox4siRNA transient transfection, the HG-induced NADPH activtion and ROS generation in MCs and cell proliferation were inhibited obviously, suggesting that the effect of HG on promoting cell growth might be closely related to Nox4upregulation and NADPH oxidase activation.3. HG induced Akt and NF-κB activation as manifested by that the relative amount of phosphorylated Akt and IκBα and degradation of IκBα are significantly higher than that of control cells. Treatment of NADPH oxidase inhibitor (DPI,10μM) or ROS inhibitor (Tempol,100mM) blocked Akt and NF-κB activation in MCs by HG, indicating that NADPH oxidase activation by HG is the upstream of Akt and NF-κB in MCs. HG-induced Akt phosphorylation was abolished by PI3K inhibitor (LY294002,10μM) treatment, whereas Sulfasalazine (Sul,500μM), an IκBα inhibitor, failed to inhibit Akt phosphorylation. Meanwhile, IκBα Phosphorylation and degradation were inhibited in the presence of Sul, LY294002, and DPI or Tempol. Futhermore, LY294002effectively inhibited HG-induced upexpression of Nox4as well as prevented by DPI. However, no significant change in expression level of Nox4was observed in MCs treated with Tempol or Sul. All together, the above results suggested that NADPH oxidase and Akt activation might be in parallel or interplay with each other, which are upstream of NF-κB pathway in the HG-stimulated signaling pathway in MCs.4. Incubation of MCs with HG for48h markedly decreased the expression levels of TRPC6protein and TRPC6mRNA compared with NG. The intracellular free calcium concentration in MCs was also decreased by HG. The down-expression of TRPC6induced by HG was obviously abrogated by Nox4or IκBα siRNA transient transfection. Furthermore, in the presence of inhibitor of signaling molecules, which are LY294002, DPI, Tempol and Sul, HG-induced TRPC6down-expression and calcium influx reduction were markedly abolished. These results suggested that HG might induce the contractile dysfunction of MCs by down-regulating the TRPC6expression and inhibiting calcium influx through NADPH oxidase/ROS/Akt/NF-κB signaling pathway.5. Administration of AS-Ⅳ in the concentration range of25-100μM showed in significant inhibition of MCs growth and hypertrophy induced by HG The downregulation of TRPC6and reduction of intracellular free calcium concentration in MCs stimulated by HG were also abrogated by AS-IV administration in a dose dependent manner.6. The levels of ROS generation, NADPH oxidase activation, Nox4upregulation, phosphorylation of Akt and IκBα, and degradation of IκBα were also obviously ameliorated by the treatment of AS-Ⅳ at concentration from25to100μM. These results suggested that AS-Ⅳ might inhibit HG-induced proliferation and reduction of calcium influx in MCs under HG conditions through inhibiting NADPH oxidase/ROS/Akt/NF-κB signaling pathway.Conclusion1. High glucose could induce glomerular MCs proliferation and downexpression of TRPC6protein by promoting Nox4upregulation, NADPH oxidase activation, ROS generation, Akt and NF-κB activation. NADPH oxidase activation and Akt activation could be in the parallel or regulatory crosstalk each other, which are upstream of NF-κB pathway in the HG-stimulated signaling pathway in MCs. 2. AS-IV treatment might prevent damage of MCs in HG through inhibiting NADPH oxidase/ROS/Akt/NF-κB signaling pathway. Therefore, we believe that AS-Ⅳ might become a valuable candidate for preventing and treating early DN. However, other relevant mechanisms underlying the effect of AS-Ⅳ require further investigations. |
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