Advanced oxidation protein products upregulate efflux transporter expression and activity through activation of the Nrf-2-mediated signaling pathway in vitro and in vivo.

Clinical and benchtop studies suggest that chronic kidney disease (CKD) alters both renal and nonrenal clearance of drugs. Although studies have documented that the accumulating uremic toxins in the body under CKD conditions are humoral factors that alter the expression and/or activity of drug transporters, the specific process is poorly understood. In this study, we found that advanced oxidation protein products (AOPPs), which are a modified protein uremic toxin, could upregulate efflux transporters, including P-glycoprotein (ABCB1), multi-drug resistance-associated protein 2 (ABCC2) and breast cancer resistance protein (ABCG2) expression in CKD rat models and in HepG2 cells. Our research shows that renal function decline was associated with the accumulation of AOPPs in serum and the upregulation of efflux transporters in the liver in two rat models of CKD. In HepG2 cells, AOPPs significantly increased the expression of efflux transporters in a dose- and time-dependent manner and upregulated the mRNA expression, protein expression and activity of efflux transporters, but bovine serum albumin (BSA), a synthetic precursor of AOPPs, had no effect. This effect correlated with AOPPs activation of the nuclear factor E2-related factor 2 (Nrf-2)-mediated signaling pathway. Further investigation of the regulation of Nrf-2 by AOPPs revealed that ML385 and siNrf-2 abolished the upregulatory effects of AOPPs. These findings suggest that AOPPs upregulate ABCB1, ABCG2 and ABCC2 through Nrf-2 signaling pathways. Protein uremic toxins, such as AOPPs, may modify the nonrenal clearance of drugs in patients with CKD through effects on drug transporters.

Protective effects of Paeoniflorin against AOPP-induced oxidative injury in HUVECs by blocking the ROS-HIF-1α/VEGF pathway.

BACKGROUND: Paeoniflorin, a monoterpene glycoside, exerts protective vascular effects, showing good antioxidant properties. However, whether Paeoniflorin has protective effect against the oxidative damage induced by advanced oxidation protein products (AOPPs) in Human umbilical vein endothelial cells (HUVECs) is unknown, as is the underlying mechanism. PURPOSE: The present study was designed to investigate the effect of Paeoniflorin on oxidative damage of HUVECs and elucidate its underlying molecular mechanisms. METHODS: The fluorescence intensity of 2', 7'-dichlorofluorescein-diacetate (DCFH-DA) staining was detected for intracellular reactive oxygen species (ROS) production. The increases mitochondrial membrane potential (MMP) was measured via flow cytometry and confocal microscopy using MitoTracker® Deep Red/ MitoTracker® Green staining. The intracellular adenosine triphosphate (ATP) was measured by ATP Determination Kit according to the manufacturer's protocol. Nox2, Nox4, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65 expressions were detected by western blot. RESULTS: Our results showed that Paeoniflorin increases MMP and ATP levels of HUVECs induced by AOPPs, and attenuates NF-κB p65 expression on HUVECs might mainly result from its antioxidant capability by suppressing ROS production. Moreover, we also found that Paeoniflorin can suppress HIF-1α and VEGF protein expression through a decrease of ROS production via down-regulation of Nox2/Nox4 expression in HUVECs. AOPP-induced RAGE mRNA up-regulation was blocked by Paeoniflorin treatment in HUVECs. CONCLUSION: Our results provided the first experimental that Paeoniflorin protects against AOPP-induced oxidative damage in HUVECs, mainly through a mechanism involving a decrease in ROS production by the inhibition of Nox2/Nox4 and RAGE expression; restored ATP depletion and mitochondria dysfunction via ROS suppression; and down-regulated HIF-1α/VEGF, possibly via the ROS-NF-κB axis.

Huang Gan Formula Eliminates the Oxidative Stress Effects of Advanced Oxidation Protein Products on the Divergent Regulation of the Expression of AGEs Receptors via the JAK2/STAT3 Pathway.

Chronic kidney disease (CKD) has a high prevalence and low cure rate and represents a significant health issue. Oxidative stress is common in CKD due to metabolic disorders, inflammation, and impaired renal function changing normal proteins into advanced oxidation protein products (AOPPs). Huang Gan formula (HGF) is a new type of traditional Chinese herbal medicine. Although we previously investigated the protective effects of HGF against oxidative stress, the mechanism of HGF in CKD is still not fully understood. In this study, we used western blotting, quantitative polymerase chain reaction, and biochemical assays to show that HGF significantly decreased AOPP-induced oxidative stress damage. Moreover, the protective effects of HGF might be associated with upregulation of the advanced glycation end product receptor 1 (AGE-R1) and downregulation of the receptor for advance glycation end products (RAGE). Treatment with HGF and the Janus kinase 2 (JAK2) inhibitor, AG4-90, significantly attenuated AOPP-induced JAK2/STAT3 protein levels. These findings indicate that HGF inhibits AOPP-mediated biological responses by inactivating the JAK2/STAT3 pathway. In conclusion, HGF eliminated AOPP-induced effects in human mesangial cells (HMCs) by interrupting JAK2/STAT3 signaling, which altered RAGE/AGE-R1 expression and reduced oxidative stress in CKD.

Advanced oxidation protein products induce chondrocyte death through a redox-dependent, poly (ADP-ribose) polymerase-1-mediated pathway.

This study aimed to investigate the effect of AOPPs on apoptosis in human chondrocytes. Chondrocytes were treated with AOPPs. Cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, reactive oxygen species (ROS) generation, and the expression of apoptotic proteins were detected in vitro. AOPPs levels were detected by colorimetric method. The results in vitro demonstrated that AOPPs induced cell death in human chondrocyte through a redox-dependent pathway, including RAGE-mediated, NADPH oxidase-dependent ROS generation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Targeting AOPPs-induced cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

Advanced oxidation protein products induce catabolic effect through oxidant-dependent activation of NF-κ B pathway in human chondrocyte.

Advanced oxidation protein products (AOPPs) have been shown to participate in the progression of rheumatoid arthritis (RA). However, the effect of AOPPs accumulation on catabolic effect in human chondrocyte and the underlying mechanism(s) remain unclear. The present study demonstrated that AOPPs inhibited cell viability and glycosaminoglycan (GAG) production in human chondrocyte. Exposure of chondrocyte to AOPPs significantly increased the production of catabolic factors, such as cyclooxygenase-2 (COX-2), matrix metalloproteinase 3 (MMPs)-3 and MMP-13. AOPPs stimulation induced ROS generation and NF-κ B p65 phosphorylation, which could be inhibited by soluble receptor for advanced glycan end products (sRAGE), NADPH oxidase inhibitor (apocynin), ROS scavenger (N-acetyl-cysteine, NAC). Furthermore, NF-κ B inhibitor Bay11-7082 significantly reversed the AOPPs-induced expression of catabolic factors and phosphorylation of NF-κ B p65. Targeting AOPPs-triggered catabolic effect might be as a promising option for patients with RA.

Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/ß-catenin signaling pathway.

Huang Gan formula (HGF) is a new traditional Chinese herbal medicine created according to the basic theory of traditional Chinese medicine. The aim of this study is to evaluate the effects of HGF on chronic kidney disease and determine the mechanisms of action. The extract of HGF was prepared, and qualitative and quantitative determination of phytochemical was performed with quadrupole time-of-flight mass spectrometer and high-performance liquid chromatography. Sprague-Dawley rats (n=72) were submitted to 5/6 nephrectomy (Nx), and then respectively treated with uremic clearance granule, losartan, HGF low dose, HGF middle dose, and HGF high dose once per day for 12 weeks. The sham group of operated rats (n=22) was treated with normal saline or HGF middle dose as a background control group. Blood and urine biochemical parameters, renal tissue morphology, and mRNA and proteins of Wnt/ß-catenin signaling pathways were investigated. The results showed that the quality of the extraction process could be controlled, and a total of eight major compounds were identified and quantified. HGF could decrease the level of serum creatinine, blood urea nitrogen, and urine protein and increase the renal index and creatinine clearance rate in a dose-dependent manner. HGF also remarkably reduced the glomerulosclerosis and tubulointerstitial fibrosis by blocking the Wnt/ß-catenin signaling pathway through inhibiting the Wnt1, ß-catenin, transcription factor 4, and fibronectin 1 expressions, simultaneously measured through mRNA and protein levels in the remnant kidney. These results suggest that extraction of HGF could improve remnant renal function and possibly ameliorate glomerulosclerosis and tubulointerstitial fibrosis by depressing the Wnt/ß-catenin signaling pathway.

[Compound Huang Gan delays chronic renal failure after 5/6 nephrectomy in rats].

OBJECTIVE: To observe the effect of compound Huang Gan in delaying chronic renal failure in rats after 5/6 nephrectomy and explore the possible mechanisms. METHODS: High-performance liquid chromatography was used to was used identify the components of compound Huang Gan extract. Rat models of 5/6 nephrectomy received a 12-week treatment with intragastric administration of Niaoduqing, Cozaar, or compound Huang Gan at low, moderate or high doses (n=10). After the treatments, the rats were sacrificed for detecting Scr, BUN, Ucr and 24h UPr , pathological examination of the renal tissues, and determination of FN, MCP-1, and ICAM-1 expression levels in the renal tissues using RT-PCR and immunohistochemistry. RESULTS: The major chemical components of compound Huang Gan extract included glycyrrhizin (0.61%), paeonol (1.2%), aloe emodin (0.72%), rhein (0.85%), emodin (0.87%), chrysophanol (0.79%) and physcion (0.8%). Treatment with compound Huang Gan at low, moderate and high doses significantly reduced Scr, BUN, Ucr , Ccr and 24 h UPr levels (P(P<0.05), improved interstitial fibrosis and glomerulosclerosis, and reduced FN and ICAM-1 expressions (P(P<0.05) in rats following nephrectomy. CONCLUSIONS: Compound Huang Gan can improve the renal function and lessen glomerulosclerosis and renal interstitial fibrosis to delay the progression of chronic renal failure in rat models of 5/6 nephrectomy.

Paeonol attenuates advanced oxidation protein product-induced oxidative stress injury in THP-1 macrophages.

BACKGROUND: Paeonol (2'-hydroxy-4'-methoxyacetophenone) is thought to possess a broad range of clinically curative effects that are likely mediated by its anti-inflammatory and antioxidant activities. AIMS: To elucidate the efficacy of paeonol's anti-inflammatory and antioxidant activities and the underlying mechanism of paeonol in advanced oxidation protein product (AOPP) stimulation of THP-1 macrophages. MATERIALS AND METHODS: After incubating cells with AOPP plus paeonol, nitric oxide (NO) production and the levels of inducible NO synthase (iNOS), receptor for advanced glycation end products (RAGE), CD36, scavenger receptor (SR)-A, and SR-B1 were calculated. Moreover, THP-1 macrophages were preincubated with paeonol, the free radical scavenger N-acetylcysteine (NAC), NADPH oxidase inhibitors [apocynin, diphenylene iodonium (DPI)], and the specific inhibitor of nuclear factor-κB pyrrolidine dithiocarbamate (PDTC) prior to incubation with AOPP, and the levels of intracellular reactive oxygen species (ROS) production and tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and monocyte chemotactic protein 1 (MCP-1) were determined. RESULTS: Paeonol increased NO production and the mRNA level of iNOS, whereas it decreased ROS production. ROS production was also effectively attenuated by apocynin, DPI, NAC, and PDTC. Furthermore, these inhibitors and paeonol could downregulate the mRNA and protein levels of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and MCP-1). Paeonol significantly reduced the expression levels of RAGE and CD36 but increased the expression levels of SR-A and SR-B1. CONCLUSIONS: These results indicate that paeonol can decrease proinflammatory cytokines in THP-1 macrophages, likely through RAGE-, CD36-, SR-A-, and SR-B1-mediated signals involving NADPH oxidase-dependent ROS generation. This suggests that paeonol can be used as a therapeutic agent for diseases contributing to oxidative stress injury.