The Effect Of Honokiol On Renal Cell Protection And Its Mechanism

Diabetes mellitus (DM) now accounts for more cases of end-stage renal disease (ESRD) than any other cause of chronic kidney disease (CKD). Glycemic control along with currently available pharmacotherapies slow, but do not stop, the progression of DN towards ESRD. Over the past few decades, mesangial cells and the GBM have tended to be the focus of many DN studies. But now many studies of diabetic patients and animal models reveal that the onset of albuminuria is most closely associated with podocytopathies, such as podocyte hypertrophy, detachment and apoptosis.Recently, more and more scholars make inflammation important in the pathogenesis of diabetic nephropathy. Renal intrinsic cells in pathological conditions can produce a variety of inflammatory factors, and then these factors expand the inflammatory response through autocrine and paracrine pathways to cause the inflammatory cascade. In normal conditions, the body can produce a small amount of reactive oxygen species (ROS) involved in the metabolism. In vivo, there is free radical scavenging reaction system which can scavenge the excessive free radicals or reduce free radical production, so that the production and clearing of ROS in a state of dynamic equilibrium. But diabetes can make this balance destruction. ROS generation increasing or ROS clearing reduction result in accumulate of ROS in the body which will lead to kidney cells damage, especially podocyte cell injury. Inflammation and oxidative stress has become the important targets for treatment of diabetic nephropathy. So finding and exploring an effective drug with not only anti-inflammatory and antioxidant role, but also no obvious side effects is particularly important.Honokiol (HNK), an active ingredient purified from Magnolia obovata, is one of the traditional medicine in Asia. With the progress research, HNK has been shown to have multiple beneficial pharmacological effects including anti-inflammatory, anti-anxiety, anti-bacterial, anti-tumor and antioxidant roles. Therefore, in the the present study, evaluation of the protective effect of HNK on inflammatory response stimulated by high glucose and LPS in renal mesangial cell and apoptosis under oxidative stress environment in glomerular epithelial cells, and investigation the underling mechanism through the detection of the changes of intracellular signaling molecules, to actively explore and find an effective therapy for diabetic nephropathy Part one:Inhibition of honokiol to the expression of inflammatory factors induced by high glucose in human mesangial cellsObjective:To evaluate the regulatory effects of honokiol on high-glucose (HG,50mmol/1)-induced inflammatory responses of Human renal mesangial cells(HRMCs).Methods:We performed MTS assays to determine the non-cytotoxic concentration of honokiol for HRMC. The impact of different concentrations of HG on the apoptosis rate was detected with Annexin V staining and follwoing flow cytometry method to determine the active concentration of HG. Enzyme-linked immunosorbent assays were per-formed to analyze the expressions of the proteins interleukin(IL)-1β, IL-18, tumor necrosis factor(TNF)-a, transforming growth factor(TGF)-β1, CCL-2, CCL-3, CCL-5and prostaglandin(PG)E2. The total nitric oxide(NO) concentration was determined using the Griess reaction.Results:Honokiol did not significantly change HRMC viability when used at a concentration of<20μmol/1but markedly altered cell viability at concentrations of>40μmol/1. Flow cytometric detection revealed that50mmol/l glucose concentration did not significantly affect the rate of early apoptosis of HRMC, which was considered as the active concentration for subsequent experiments. Treatment with50mmol/L glucose markedly increased the level of IL-1β, IL-18, TNF-a, PGE2, NO, TGF-β1, CCL-2, CCL-3and CCL-5. Honokiol (0-20μmol/1) treatment inhibited the HG-induced expression of inflammatory cytokines such as IL-1β, IL-18, TNF-a, PGE2, NO, and TGF-β1in a dose-dependent manner. Moreover, it markedly inhibited the expression of Chemokines such as CCL-2, CCL-3and CCL-5, which are upregulated under HG conditions. Conclusion:Honokiol inhibits the HG-induced expression of inflammatory factors in HRMCs. Honokiol may be considered a promising drug with potent anti-inflammatory activities in addition to its strong anti-cancer, anti-angiogenesis, and anti-neuro degenerative effects. Part two:Inhibitory effects of honokiol on lipopolysaccharide-induced cellular responses and signaling events in human renal mesangial cellsObjective:To investigate the anti-inflammatory effects of honokiol and the signaling mechanisms involved in lipopolysaccharide (LPS)-induced conditions in human renal mesangial cells (HRMCs).Methods:MTS detection the proliferation rate of the HRMC treated with different concentrations LPS was in order to determine the toxic concentrations of LPS on HRMC. Enzyme-linked immunosorbent assay(ELISA) preliminary was performed to test the effect on the levels of.TNF-α and CCL-2protein expression in different concentrations of LPS in the HRMC. Then MTS result was combined with ELISA results to choose the LPS concentration to continue our study. Enzyme-linked immunosorbent assays were per-formed to analyze the expressions of the proteins IL-1β, IL-18, TNF-a, TGF-β1, CCL-2, CCL-3, CCL-5and PGE2. The total NO concentration was determined using the Griess reaction. Phospho-NF-KB p65, phospho-IKK-alpha/beta, IKB-alpha, phospho-Akt, phospho-p42/44MAPK and their non-phospho-proteins, COX-2and iNOS were determined with western blot.Results:LPS did not significantly change HRMC viability when used at a concentration of<2μg/ml but altered cell viability at concentrations of>5μg/ml. With the increase of LPS concentration(0-1μg/ml), the expressions of TNF-aand CCL-2protein gradually increased. But there is no significant difference of TNF-aand CCL-2protein expressions when LPS concentration is between1μg/ml and2μg/ml. In this study, LPS treatment led to a marked upregulation of the levels of IL-1β, IL-18, TNF-β, TGF-β1, CCL-2, CCL-3, and CCL-5in HRMCs. The expression of COX-2, iNOS, and their products PGE2and NO also increased. The upregulation of these molecules was significantly abolished by honokiol in a dose-dependent manner. Moreover, honokiol almost completely reversed IL-1β, CCL-3, and NO expression at10μmol/1, and IL-18, TNF-a, TGF-β1, and COX-2expression at20μmol/l. In addition, phospho-NF-KB p65, phospho-IKK-alpha/beta, IKB-alpha, phospho-Akt, and phospho-p42/44MAPK were dramatically suppressed by honokiol in LPS-treated HRMCs.Conclusion:These results indicate that honokiol can inhibit the LPS-induced expression of inflammatory cytokines and mediators in HRMCs. The anti-inflammatory mechanisms of honokiol are partly due to the suppression of the phospho-NF-KB p65, phospho-Akt, phospho-p42/44, COX-2and iNOS pathways. Part three:Protective effects of honokiol on oxidative stress induced cell apoptosis and signaling events in mouse podocyteObjective:Oxidative stress is a common factor in the renal tissue damage caused by diabetes or other diseases. It also contributed to one of the important reasons of podocyte apoptosis. In the present study, we observed the effect of HNK on cultured mouse podocytes apoptosis induced by H2O2. The expressions of part of the intracellular signaling molecules were dectected to investigate the underlying mechanism of the protective role of HNK.Methods:Cultured mouse podocytes were treated with different concentrations of HNK and100μM H2O2:First, the number of floating cells in the supernatant and the general state of adherent cells were observed under inverted phase contrast microscope. MTS assay and flow cytometry method were performed for detection of podocyte cell proliferation activity and apoptotic rate. The change of the apoptotic protein caspase-3and caspase-9were investigated with real-time quantitative polymerase chain reaction and immune blotting. In addition, Erkl/2and Akt signaling molecule phosphorylation levels were determined with the western blot in order to further investigate the mechanism of the antioxidant role of HNK.Results:The deterioration of cell state was observed under light microscope after24h cultured with H2O2, and there were the large amounts of visible black specks in the podocytes which may be the swelling of organelles. MTS assay and Annexin V/PI double staining further confirmed that H2O2can lead to the decline of the proliferation rate in podocytes and significantly increasing of apoptotic rate. After24h treated with different concentrations of HNK, and with the concentration increasing, the status of adherent podocytes were improved and gradually become transparent. And with the dead cells floating in the supernatant decreased, the rate of cell proliferation was increased and apoptosis was significantly decreased. Real-time PCR and western immunoblotting results showed that HNK significantly downregulated the increased expression of cleaved protein and mRNA of caspase-3and caspase-9expression in podocytes stimulated by H2O2. Moreover, the levels of phosphorylation of Erkl/2and Akt signaling molecules were no significant decline, but seems to increase to some extent after24h stimulated by H2O2. And HNK in low, medium and high concentrations further upregulated the phosphorylation levels of Erkl/2and Akt.Conclusion:HNK largely eliminated the role of promoting podocyte apoptosis by oxidative stress environment which was a protective factor on cultured podocytes with H2O2. In addition, it was an important mechanism for HNK to the reduction of podocyte apoptosis that HNK significantly decresed the expressions of active protein and mRNA of caspase-3and caspase-9. The level of phosphorylation of Erkl/2and Akt signaling molecules further increased by HNK may also be one of the mechanisms. But we still need to detect the response curve of time-proteins phosphorylation to further investigate the changing rule of Erkl/2and Akt.