The purpose of our study is to clarify the role of KMP in the progression of DN. The results suggest that KMP may have therapeutic potential in DN. The effective concentration of KMP was determined by MTS method. The treatment of KMP not only decreased ROS generation via increasing anti-oxidative stress proteins, but also decreased apoptosis-related proteins by modulating ROS-sensitive Akt/FoxO signaling pathway.
KMP, an important flavonoid, has been reported to exist in plenty of plants. Accumulating studies suggest that KMP has a protective effect against age-related diseases through exerting its effects of anti-oxidation, anti-apoptosis, anti-inflammation and so on. The therapeutic effect of KMP on DN has been studied in this research. Firstly, the safe and effective concentration of KMP in our experiment was identified by MTS assays. Previous studies suggest that KMP possesses contradictory pharmacological effects. The anti-oxidant or pro-oxidant activity of KMP depends on its concentration. It has been reported that 50µM KMP has pro-oxidant and pro-apoptosis effects in glioblastoma cell, human non-small cell lung carcinoma cell line and human chronic myelogenous leukemia cell[24, 25, 26]. The MTS results in our study suggested that 50µM KMP could decrease the viability of HK-2 cells significantly. Nevertheless, 10µM or 20µM KMP treatment could markedly reverse HG-induced inhibition of cell viability. The results indicated that the effects of KMP were significantly concentration-dependent and were most notable after 48h. To avoid the cytotoxic effect and to get the greatest biological effect, we selected the concentration of 10µM KMP in the following experiments.
Accumulating researches demonstrate that KMP could ameliorate insulin resistance in diabetes[18], protect against doxorubicin-induced and isoproterenol-induced cardiotoxicity[27, 28], and protect SH-SY5Y cells and primary neurons from rotenone toxicity[29] owing to KMP-regulated anti-apoptotic and anti-oxidant effects. It has been well established that excessive generation of mitochondrial ROS induced by HG is the primary initiating mechanism in the development and progression of DN. What's more, accumulating evidence suggests that renal tubular cell apoptosis is associated with the pathogenesis of DN[30]. Hence, finding compounds with potential anti-oxidative and anti-apoptotic property is necessary to counterwork hyperglycemia-caused kidney injuries. In our study, HG obviously increased oxidative stress and apoptosis-related proteins. The treatment of 10µM KMP in HK-2 cells significantly prevented HG-induced overproduction of ROS and depletion of anti-oxidants proteins, SOD2 and catalase. Furthermore, Western blotting results revealed that KMP treatment for 48h could increase Bcl-2 expression and decrease the proapoptotic proteins, Bax and cleaved caspase-3, indicating the decreased level of apoptosis in HK-2 cells. Taken together, KMP could counteract HG-induced injuries in HK-2 cells with its anti-oxidative and anti-apoptotic effects.
The major molecular mechanisms responsible for the KMP-mediated anti-oxidant effect have not been elucidated completely. Sirt3, a protein deacetylase preferentially localized in mitochondria, mediates the deacetylation of enzymes responsible for generating ROS and plays a role in several metabolic processes[31]. In addition, it has been reported that the overexpression of Sirt3 could upregulate the expression and activity of SOD2 and catalase in cardiomyocytes[32]. Furthermore, our recent study demonstrates that Sirt3 overexpression could significantly attenuate HG-induced oxidative stress and apoptosis in HK-2 cells[33]. In general, Sirt3 plays a critical role in protecting against ROS generation. Recently, it has been suggested that KMP could increase the expression of Sirt3 in chronic myelogenous leukemia cells[23, 26]. Thus, we hypothesized that Sirt3 might contribute to the anti-oxidative effect of KMP. The obtained results showed that the treatment of KMP markedly reversed the protein and mRNA expression of Sirt3 under HG stimulation. It suggested that KMP exerted its anti-oxidant effect via increasing Sirt3 expression.
Forkhead box O3a (FoxO3a) plays a vital role in regulating cell survival, oxidative stress and apoptosis. Phosphorylation of FoxO3a by serine/threonine kinase Akt results in nucleus exclusion and cytoplasm relocalization. As a result, the upregulation of p-FoxO3a could decrease the transcription of apoptosis-related proteins, Bim, FasL and p27[34]. Our previous research indicates that Sirt3 could regulate cell apoptosis via ROS-sensitive Akt/FoxO signaling pathway in HK-2 cells[33]. In this experiment, the data showed that KMP observably reversed the downtrend of Sirt3 expression in HG. Thus, we speculated that KMP could alter the activity of Akt/FoxO signaling pathway. The Western blot experiment showed that 10µM KMP dramatically increased the ratio of p-Akt/Akt and p-FoxO3a/FoxO3a, leading to the decrease of transcriptional activity of FoxO3a and the expression of pro-apoptotic proteins.
In conclusion, the data in our experiment firstly demonstrate that KMP may exert its anti-oxidative and anti-apoptotic effects through increasing the expression of Sirt3 and regulating ROS-sensitive Akt/FoxO signaling pathway. Our research suggests that KMP may have therapeutic effects for DN.