A method for screening nephroprotective compounds in cortex Moutan, a common traditional Chinese medicine (TCM) in treating diabetic nephropathy with renal mesangial cell extraction and ultra performance liquid chromatography technique was described in this paper. We hypothesize that the compounds which bind to cell membranes under pathological conditions may be the bioactive compounds in TCMs. Mesangial cells were cultured in medium containing 5 mM (physiological, NG) or 30 mM (pathological, HG) glucose for 48 h and then incubated with cortex Moutan extract. After the unbound substances were washed off, the cell membrane-bound compounds were dissociated and concentrated by an SPE column. By comparing the chromatograms of NG and HG cultured-cell extractions and cortex Moutan extract, three compounds bound to both NG and HG-cultured mesangial cells were identified as paeoniflorin, pentagalloylglucose (PGG) and paeonol. In vitro studies showed that paeoniflorin, PGG and paeonol reduced the activity of nicotinamide-adenine dinucleotide phosphate oxidase (NADPH) activity, and decreased the level of reactive oxygen species (ROS), transforming growth factor-β1 (TGF-β1) and fibronectin in high glucose cultured mesangial cells. The results indicate that paeonol, paeoniflorin and PGG may be the nephroprotective compounds from cortex Moutan. This study is expected to provide a more reliable and effective method for screening bioactive compounds from the complex TCM systems.
The antidiabetic properties and anti-inflammatory effects of Danzhi Jiangtang Capsules (DJC) have been demonstrated in clinical and laboratory experiments. In this study, we explored whether DJC can ameliorate advanced glycation end products- (AGEs-) mediated cell injury and the precise mechanisms of DJC in treating diabetic nephropathy (DN). Western blot analysis was employed to assess the expressions of iNOS, COX2, and SOCS and the phosphorylation of JAK2, STAT1, and STAT3 in glomerular mesangial cells (GMCs) after treatment with DJC. TNF-α, IL-6, and MCP-1 were determined using double-antibody sandwich ELISA. ROS and NADPH oxidase activity were measured by DCFH-DA assay and lucigenin-enhanced chemiluminescence, respectively. DJC significantly reversed the AGEs-induced expression of COX2 and iNOS. Moreover, DJC inhibited the AGEs-induced JAK2-STAT1/STAT3 activation, resulting in the inhibition of inflammatory cytokines such as IL-6, MCP-1, and TNF-α in a concentration-dependent manner. The ability of DJC to suppress STAT activation was also verified by the observation that DJC significantly increased the SOCS3 protein level. DJC reversed the AGEs-induced accumulation of ROS and NADPH oxidase activity, thus confirming that DJC possesses antioxidant activity. The results suggest that the anti-inflammatory effects of DJC in GMCs may be due to its ability to suppress the JAK2-STAT1/STAT3 cascades and reduce ROS production.
Danzhi Jiangtang Capsule (DJC), a traditional Chinese medicinal formula, has been used clinically in treating diabetes and diabetic nephropathy (DN). We previously demonstrated that DJC is capable of improving renal function in patients and rats with DN, but the mechanisms underlying these therapeutic benefits of DJC are not quite clear yet. In this study, STZ-induced diabetic rats were orally administered DJC for 8 weeks. Fasting blood glucose, renal function indicators in the serum, renal index, and the expression of proteins related to JAK-STAT signaling pathway were evaluated at the end of the experiment. The kidneys were sliced for pathological histology. Antioxidant status was assessed by measuring SOD, LPO and MDA in serum. The expression levels of COX2, iNOS, SOCS and the phosphorylation status of JAK2, STAT1, and STAT3 in renal tissues were evaluated by Western blot analyses. IL-6, TNF-α, and MCP-1 expression levels in renal tissues were determined using doubleantibody sandwich ELISA. Diabetic renal dysfunction and its associated pathologies were ameliorated by DJC treatment. DJC significantly reversed the high expression of COX2 and iNOS in renal tissues. Furthermore, DJC inhibited the JAK2-STAT1/STAT3-SOCS3 signaling pathway, resulting in decreased concentrations of IL-6, TNF-α, and MCP-1. Moreover, the oxidant status in the kidney was substantially ameliorated by DJC treatment. In conclusion, the ability of DJC to ameliorate diabetic renal dysfunction and the associated pathologies of this disease might be due to its antioxidant capacity and suppression of the JAK2-STAT1/ STAT3 cascade.
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