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ErHuang Formula Improves Renal Fibrosis in Diabetic Nephropathy Rats by Inhibiting CXCL6/JAK/STAT3 Signaling Pathway.

ABSTRACT: Diabetic nephropathy (DN) is one of the main causes of renal fibrosis and is associated with high morbidity and mortality. Traditional Chinese Medicine (TCM) therapy has a long history of usage in a clinical setting and its usage is increasing. ErHuang Formula (EHF), a Chinese herbal compound, has been clinically used in treating DN for more than 30 years. However, its mechanism of action is still unknown. This study was conducted to evaluate the effect of EHF on renal fibrosis in a DN rat model and explore its underlying mechanism. The DN rat model was established by high-sugar-fat diet combined with a single intraperitoneal injection of streptozotocin (STZ), and EFH extract (4, 2, 1 g/kg d-1) was administered orally for 8 weeks. The biochemical parameters (blood glucose, weight, Scr, BUN, UA, U-Alb and UAE) were analyzed. The pathological changes in renal tissue were observed by histological staining with H&E and Masson. The effect of EHF on the proliferation of NRK-49F cells was examined by CCK-8 assay and the levels of several inflammation and fibrosis related cytokines (IL-6, TNF-α, TGF-β1, Collagen I/III, MMP2/9) in serum and NRK-49F cell culture supernatants were detected by enzyme-linked immunoassay (ELISA). The mRNA levels of CXCL6, CXCR1, Collagen I/III, MMP2/9 in renal tissue were also measured by quantitative RT-PCR. Furthermore, the protein expression of PCNA, Collagen I/III, MMP2/9, CXCL6, CXCR1, p-STAT3, STAT3 in renal tissue and NRK-49F cells were determined by western blot. EHF improved the abnormal biochemical parameters and ameliorated the abnormal histology and fibrosis of renal tissue in a dose-dependent manner. EHF inhibited NRK-49F proliferation and decreased the expressions of inflammation and fibrosis related factors both in vitro and in vivo. Interestingly, the levels of Collagen I/III, PCNA, MMP2/9 and p-STAT3 were positively correlated with CXCL6. The amelioration of renal fibrosis in DN by EHF is related to CXCL6/JAK/STAT3 signal pathway, which is associated with inflammation and fibrosis of the tissue. These findings may have clinical implications for the treatment of DN.

SUBMITTER: Shen YL

PROVIDER: S-EPMC6993046 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMorus alba L. (Sangzhi) alkaloid (SZ-A) is a new antidiabetic drug approved by the China National Medical Products Administration in 2020. Diabetic nephropathy (DN) is a common diabetic complication and an important cause of morbidity and mortality in patients with diabetes. The effects of SZ-A on DN remain unknown.PurposeThis study evaluated the effects of SZ-A on DN in Zucker diabetic fatty (ZDF) rats and explored the underlying mechanisms based on nitrosative stress, inflammation, and fibrosis.MethodsDiabetic ZDF rats were orally administered 100 and 200 mg/kg of SZ-A once daily for 9 weeks. The glucose metabolism and kidney function were assayed. The pathological injury and fibrosis of the kidneys were separately evaluated using hematoxylin and eosin staining and Masson's staining. The oxidative and nitrosative stress and inflammation were assayed by determining the levels of related indices in the blood and kidneys and quantifying the related gene and protein expression. The expression of transforming growth factor β1 (TGFβ1) gene and protein were assayed by quantitative real-time PCR and immunohistochemistry, respectively. The renal transcriptomics was analyzed using RNA sequencing.ResultsRepeated treatment with SZ-A significantly improved glucose metabolism, dose-dependently decreased the levels of blood urea nitrogen, urinary albumin, and β2-microglobulin, and evidently relieved the renal injury in diabetic ZDF rats. As for the mechanisms, SZ-A remarkably ameliorated systemic nitrosative stress through lowering the levels of blood inducible nitric oxide synthase and nitric oxide, and significantly relieved systemic and renal inflammation by reducing the levels of blood interleukin-1β and monocyte chemoattractant protein-1 (MCP-1) and decreasing the levels of renal C-reactive protein content and expression of tumor necrosis factor-α in the kidneys. SZ-A also improved renal fibrosis by lowering the expression of TGFβ1 in the kidneys. Additionally, SZ-A significantly lowered the expression of stimulator of chondrogenesis 1 in the kidneys.ConclusionRepeated treatments with SZ-A significantly ameliorates DN by regulating systemic nitrosative stress, renal inflammation, and renal fibrosis partially through inhibition of the cytokine-NO and TGF-β1 signaling in ZDF rats, providing evidence for the additional application of SZ-A in clinical use for the treatment of DN.

Project description:BackgroundDiabetic nephropathy (DN) is one of the main causes of end-stage renal disease with scantly effective treatment. Numerous evidences indicated that macrophages play an important role in the occurrence and pathogenesis of DN by secreting inflammatory cytokines. Mincle is mainly expressed in macrophages and promotes kidney inflammation and damage of acute kidney injury. However, the role of Mincle in DN is unclear. In this study, we aim to investigate the effect of Mincle-related macrophage inflammation on DN, and whether it can be identified as the therapeutic target for Astragalus mongholicus Bunge and Panax notoginseng Formula (A&P), a widely used Chinese herbal decoction for DN treatment.MethodsIn vivo experiments high-fat and high-sugar diet and streptozotocin was used to establish a diabetic nephropathy model, while in vitro experiments inflammation model was induced by high-glucose in mouse Bone Marrow-Derived Macrophages (BMDM) cells and mouse mesangial (MES) cells. Kidney pathological staining is used to detect kidney tissue damage and inflammation, Western blotting, Real-time PCR and ELISA are performed to detect Mincle signaling pathway related proteins and inflammatory cytokines.ResultsMincle was mainly expressed in infiltrated macrophage of DN kidney, and was significant decreased after A&P administration. The in vitro experiments also proved that A&P effectively down-regulated the expression of Mincle in macrophage stimulated by high glucose. Meanwhile, the data demonstrated that A&P can reduce the activation of NFκB, and the expression and secretion of inflammatory cytokines in DN kidney or BMDM cells. Notably, we set up a co-culture system to conform that BMDM cells can aggravate the inflammatory response of mesangial (MES) cells under high glucose stimulation. Furthermore, we found that the anti-injury role of A&P in MES cells was dependent on inhibition of the Mincle in macrophage.ConclusionIn summary, our study found that A&P is effective in reducing renal pathological damage and improving renal function and inflammation in diabetic nephropathy by a mechanism mainly related to the inhibition of the Mincle/Card9/NFκB signaling pathway.

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ErHuang Formula Improves Renal Fibrosis in Diabetic Nephropathy Rats by Inhibiting CXCL6/JAK/STAT3 Signaling Pathway.

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