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Research ArticleInflammation, Immunopharmacology, and Asthma

Inhibition of Mitogen-Activated Protein Kinases/Nuclear Factor κB–Dependent Inflammation by a Novel Chalcone Protects the Kidney from High Fat Diet–Induced Injuries in Mice

Qilu Fang, Liancheng Deng, Lintao Wang, Yali Zhang, Qiaoyou Weng, Haimin Yin, Yong Pan, Chao Tong, Jingying Wang and Guang Liang
Journal of Pharmacology and Experimental Therapeutics November 2015, 355 (2) 235-246; DOI: https://doi.org/10.1124/jpet.115.226860

Abstract

The prevalence of obesity has increased dramatically worldwide leading to increases in obesity-related complications, such as obesity-related glomerulopathy (ORG). Obesity is a state of chronic, low-grade inflammation, and increased inflammation in the adipose and kidney tissues has been shown to promote the progression of renal damage in obesity. Current therapeutic options for ORG are fairly limited and, as a result, we are seeing increased rates of progression to end-stage renal disease. Chalcones are a class of naturally occurring compounds with various pharmacological properties. 1-(3,4-Dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one (L2H17) is a chalcone that we have previously synthesized and found capable of inhibiting the lipopolysaccharide-induced inflammatory response in macrophages. In this study, we investigated L2H17’s effect on obesity-induced renal injury using palmitic acid–induced mouse peritoneal macrophages and high fat diet–fed mice. Our results indicate that L2H17 protects against renal injury through the inhibition of the mitogen-activated protein kinase/nuclear factor κB pathways significantly by decreasing the expression of proinflammatory cytokines and cell adhesion molecules and improving kidney histology and pathology. These findings lead us to believe that L2H17, as an anti-inflammatory agent, can be a potential therapeutic option in treating ORG.

Introduction

Obesity has risen to epidemic proportions, becoming a major global health problem. An increase in body weight has been directly associated with an increased risk in developing chronic disorders, such as hypertension, hyperlipidemia, diabetes mellitus, and cardiovascular disease (Mathieu et al., 2010; Gregor and Hotamisligil, 2011; Zeng et al., 2015). All of these can promote chronic kidney disease (CKD) and—as the prevalence of obesity is rapidly increasing—the incidence of obesity-related glomerulopathy (ORG) in obese patients has also been on the rise in the population (Kambham et al., 2001; Bello et al., 2005). Although the link between obesity and nephrotic syndrome was first reported in a 1974 study, multiple studies have demonstrated diets high in fat can lead to glomerular structural and functional changes (Weisinger et al., 1974; Wei et al., 2004). However, despite the growing prevalence of ORG and how ORG is an increasing cause for end-stage renal disease (Praga et al., 2001), the therapeutic options are still very limited to mostly blood pressure and glycemic control, and there is a lack of sufficient evidence to suggest that weight loss—the logical therapeutic option for ORG—exhibits a relationship with disease progression (Tsuboi et al., 2013). These statistics cite the importance of finding and developing new therapeutic options for the treatment of obesity-related kidney disease.

An increasing number of clinical and animal studies have implicated the activation of the innate immune system and the inflammatory response in the pathogenesis of kidney disease. It is also now generally accepted that obesity is associated with systemic, low-grade inflammation (Praga et al., 2001; Praga, 2002; Mathieu et al., 2010). Circulating free fatty acids (FFAs) associated with obesity, such as palmitic acid (PA), can cause chronic inflammation, oxidative stress, insulin resistance, and cardiovascular disease as well as increase the expression of proinflammatory cytokines (Mathieu et al., 2010; Boden, 2011; Gregor and Hotamisligil, 2011). PA, which is also the most common FFA found in animals (Gunstone et al., 1994), has also been implicated in the pathogenesis of glomerulopathy and tubulointerstitial lesions in type 2 diabetes (Nosadini and Tonolo, 2011). It has also been demonstrated both in vitro and in vivo that FFAs can activate both the nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways, subsequently increasing the expression of proinflammatory cytokines, such as tumor necrosis factor (TNF) α, interleukin (IL) β, IL-1β, and interferon (IFN) γ as well as proadhesion genes, such as intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (Yamagishi et al., 2002; Boden et al., 2005; Bost et al., 2005; Nguyen et al., 2006; Zeng et al., 2015). Furthermore, there is also increasing evidence suggesting that chronically elevated FFA levels in obese individuals were responsible for the systemic inflammation observed in CKD, and the accumulation of lipids in nonadipose tissue is associated with structural and functional changes of various kidney cells, which also contributes to the development of obesity-induced renal disease (de Vries et al., 2014). Therefore, FFAs serve as a link between obesity, inflammation, and CKD, and inflammation may serve as a potential therapeutic target for the treatment of ORG.

Chalcones, which are found in a variety of plant species, are a group of compounds that belong to the flavonoid family. They have recently gained interest as studies have shown chalcones to have a variety of pharmacological activities, such as antioxidant, antihyperglycemic, antitumor, and anti-inflammatory (Shukla et al., 2007; Wu et al., 2011; Ozdemir et al., 2015; Wang et al., 2015). Chalcones can also be prepared synthetically, and previously, we have synthesized and evaluated a number of chalcone derivatives for their anti-inflammatory activities (Wu et al., 2011). Of these, 1-(3,4-dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one (L2H17) (Fig. 1A) was one of a few found to inhibit lipopolysaccharide (LPS)-induced overexpression of TNF-α and IL-6 in macrophages. Other studies done by our laboratory have also supported L2H17’s anti-inflammatory effects. However, whether L2H17 can mitigate obesity-related complications, particularly obesity-related kidney injury, remains unclear. Therefore, in this study, we employed high fat diet (HFD)-fed mice and PA-induced mouse peritoneal macrophages (MPMs) as models for obesity to explore L2H17’s effect on obesity-induced inflammation in relation to ORG. Furthermore, because other studies have connected the anti-inflammatory properties of chalcones to the NF-κB and MAPK pathways (Zuo et al., 2012; Fang et al., 2015), we also analyzed the expression of key proteins in these pathways to also elucidate the mechanism behind L2H17’s observed anti-inflammatory effects.

Fig. 1.
Fig. 1.

L2H17 inhibited the PA-induced inflammatory response in MPMs. (A) Chemical structure of chalcone and L2H17. (B–I) MPMs were pretreated with L2H17 at various doses (2.5, 5, or 10 μM), curcumin (10 μM), or vehicle [dimethylsulfoxide (DMSO) 3 μl] for 1 hour prior to stimulation with PA (100 μM) for different times. Enzyme-linked immunosorbent assay was used to detect the protein levels of TNF-α and IL-6 within the medium (for 24-hour treatment) (B and C). Total mRNA was extracted from the cell using TRIzol, and the mRNA levels of TNF-α (D), IL-6 (E), IL-1β (F), IL-2 (G), IL-12 (H), and IFN-γ (I) were detected by real-time qPCR analysis (for 6-hour treatment) (D–I). Curcumin at 10 μM was used as a comparison control. Bars represent the mean ± S.E.M. of four independent experiments performed in duplicate (*P < 0.05, **P < 0.01, and ***P < 0.001 versus the PA group).

Materials and Methods

Reagents and Cell Culture.

Palmitic acid and curcumin were purchased from Sigma (St. Louis, MO). Compound L2H17 was synthesized and characterized in our laboratory, with a purity of 98.9% (Wu et al., 2011). L2H17 was dissolved in dimethylsulfoxide for in vitro experiments and 1% sodium carboxyl methyl cellulose for in vivo experiments. Antibodies for glyceraldehyde-3-phosphate dehydrogenase, phosphorylated p65, ICAM-1, VCAM-1, inhibitor of NF-κB (IκB) α, extracellular signal-related kinase (ERK), phosphorylated ERK, and cluster of differentiation 68 were purchased from Santa Cruz (Santa Cruz, CA); and antibodies for c-Jun N-terminal kinase (JNK), phosphorylated JNK, p38, phosphorylated p38, and TNF-α were obtained from Cell Signaling (Danvers, MA). MPMs were obtained from the C57BL/6 mice and cultured in 1640 medium (Gibco, Eggenstein, Germany) with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 mg/ml of streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. Renal tubular epithelial SV40 cells (American Type Culture Collection, Manassas, VA) were seeded and grown in DMEM (Gibco) with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO2 atmosphere.

Animals.

Male C57BL/6 mice weighing 18–22 g were obtained from the Animal Center of Wenzhou Medical College (Wenzhou, China). Animals were housed at a constant room temperature with a 12-hour light/dark cycle and fed with a standard rodent diet and water. The animals were acclimatized to the laboratory for at least 3 days prior to use. All animal care and experimental procedures complied with the The Detailed Rules and Regulations of Medical Animal Experiments Administration and Implementation (document number 1998-55, Ministry of Public Health, Beijing, P.R. China), and were approved by the Wenzhou Medical College Animal Policy and Welfare Committee (approval document number 2012/APWC/0114).

Determination of TNF-α and IL-6 by Enzyme-Linked Immunosorbent Assay.

The TNF-α and IL-6 levels in the cell medium were determined with an enzyme-linked immunosorbent assay kit (Bioscience, San Diego, CA) according to the manufacturer’s instructions. The total amounts of TNF-α and IL-6 in the cell medium were normalized to the total amount of protein in the viable cell pellets.

Real-Time Quantitative Polymerase Chain Reaction.

Cells or kidney tissues (50–100 mg) were homogenized in TRIzol (Invitrogen, Carlsbad, CA) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative polymerase chain reaction (PCR) were carried out using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). The Eppendorf Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany) was used for quantitative PCR (qPCR) analysis. The primers of genes, including TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1, IL-12, IL-2, IFN-γ, IL-4, IL-10, vimentin, tumor growth factor β, collagen I, α–smooth muscle actin, monocyte chemotactic protein (MCP) 1, C-C motif chemokine receptor 2 (CCR2), and β-actin were obtained from Invitrogen (Shanghai, China). The primer sequences are listed in Table 1. The amount of each gene was determined and normalized to the amount of β-actin.

View this table:
TABLE 1

Primer sequences for real-time quantitative PCR

Western Immunoblot Analysis.

Collected cells or homogenated kidney tissue samples were lysated. Lysates (50–100 μg) were separated by 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Each membrane was preincubated for 1.5 hours at room temperature in Tris-buffered saline, pH 7.6, containing 0.05% Tween 20 and 5% nonfat milk. Then, the membrane was incubated with specific antibodies. Immunoreactive bands were detected by incubating with secondary antibodies, including goat anti-rabbit IgG–horseradish peroxidase (HRP) (Santa Cruz sc-2004), goat anti-mouse IgG-HRP (Santa Cruz sc-2458), and rabbit anti-goat IgG-HRP (Santa Cruz sc-2768), respectively, and visualizing using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA). The amounts of each protein were analyzed using Image J analysis software version 1.38e and normalized to their respective control.

Preparation of Nuclear Extracts.

Nuclear protein extraction from MPMs was done by using a nuclear protein extraction kit (Beyotime Biotech, Nantong, China) following the manufacturer’s instructions. The protein concentration was determined using the Bio-Rad protein assay reagent. The nuclear extract (15 μg of protein) was used for the Western immunoblot analysis.

Immunofluorescence for p65.

MPMs were prepared for immunofluorescence. Following treatment with the respective drug and PA, MPMs were fixed with 4% paraformaldehyde for 10 minutes and 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 minutes. Cells were incubated overnight at 4°C with anti-p65 antibody (1:200) and then incubated with phycoerythrin-labeled secondary antibody (1:400; Santa Cruz) for 1 hour. The nucleus was then incubated with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride for 5 minutes before the images were viewed under fluorescence microscope (200× amplification; Nikon, Tokyo, Japan).

Electrophoretic Mobility Shift Assay.

Nuclear protein extraction from MPMs was done as described above. Electrophoretic mobility shift assay (EMSA) was done using the EMSA kit (Beyotime Biotech). The extracted proteins were mixed with the binding buffer for 10 minutes and then mixed with the NF-κB probe for 20 minutes. The mixture was separated on a nondenaturing gel and electrotransferred onto a nylon membrane with a positive charge. Then, the membrane was preincubated with the prior blocking solution, washed with washing buffer, exposed to the detection solution, and visualized using enhanced chemiluminescence reagents (Bio-Rad).

Animal Experiments.

Curcumin was used as a positive control. To induce obesity, mice were fed a high fat diet for 8 weeks, whereas the control animals received a normal chow diet. Then, the HFD-fed mice were divided into three groups: HFD (n = 8), curcumin-treated HFD (HFD+Cur, n = 8), and L2H17-treated HFD (HFD+L2H17, n = 8). In the drug-treated groups, mice were orally administrated with curcumin at 50 mg/kg and L2H17 at 20 mg/kg every day. The HFD group and control group (n = 8) received 1% sodium carboxyl methyl cellulose solution alone according to the same schedule. Body weight was recorded weekly after drug administration. After 8 weeks of treatment, animals were sacrificed under ether anesthesia, and the blood and kidney samples were collected. Kidney tissues were embedded in 4% paraformaldehyde for pathologic analysis and/or snap frozen in liquid nitrogen for gene and protein expression analysis.

Histopathology.

Kidneys were fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned at 5 µm. After dehydration, sections were stained with hematoxylin and eosin (H&E). To evaluate the histopathological damage, each image of the sections was examined under a light microscope (400× amplification; Nikon).

Masson and Sirius Red Staining.

Tissues were fixed in paraformaldehyde solution and embedded in paraffin. Paraffin sections (5 µm) of the renal tissues were stained with the Masson trichrome staining kit (Beyotime Biotech) and Sirius red solution (Beyotime Biotech), respectively, according to the manufacturer’s instructions. The stained sections were viewed under a light microscope (400×; Nikon).

Immunohistochemistry for CD68 Detection.

After deparaffinization and rehydration, 5-µm kidney sections were treated with 3% H2O2 for 10 minutes and with 1% BSA in PBS for 30 minutes. Slides were incubated overnight at 4°C with anti-CD68 antibody (1:200) and then incubated with secondary antibody for 1 hour at room temperature. After staining the nucleus with hematoxylin for 5 minutes, the images were viewed under a light microscope (400× amplification; Nikon).

Immunofluorescence for TNF-α Detection.

After deparaffinization and rehydration, 5-µm kidney sections were treated with 3% H2O2 for 10 minutes and with 1% BSA in PBS for 30 minutes. Slides were incubated overnight at 4°C with anti–TNF-α antibody (1:300) and then incubated with phycoerythrin-labeled secondary antibody (1:200; Santa Cruz) for 1 hour at room temperature. After staining the nucleus with DAPI for 5 minutes, the images were viewed under a fluorescence microscope (400× amplification; Nikon).

Measurements of the Level of the Serum Lipid.

The components of the serum lipid, including total triglyceride (TG), total cholesterol (TCH), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), were detected using commercial kits (Nanjing Jiancheng, Jiangsu, China).

Statistical Analysis.

Data were analyzed using a t test and analysis of variance (ANOVA) in GraphPad Prism-5 statistic software (La Jolla, CA). All of the values are presented as the mean ± S.E.M. A difference of P < 0.05 was considered to be statistically significant.

Results

L2H17 Dose-Dependently Reduced the Production of an Inflammatory Response in PA-Stimulated MPMs.

PA is the most abundant and widespread FFA found in plants and animals and is usually used as a representative FFA to stimulate cellular stress response (Gunstone et al., 1994). Therefore, we first examined whether L2H17 could attenuate the PA-induced inflammatory response in MPMs. As shown in Fig. 1, PA induced an increased production of the inflammatory cytokines TNF-α and IL-6 in MPMs, while L2H17 dose-dependently inhibited TNF-α (Fig. 1B) and IL-6 (Fig. 1C) production. MPMs were first pretreated with L2H17 (2.5, 5, or 10 μM) or vehicle control (dimethylsulfoxide 3 μl) for 1 hour and then incubated with PA (100 μM) for 24 hours. Curcumin 10 μM was used as a comparative control. As shown in Fig. 1, aside from reducing PA-induced increases in protein TNF-α and IL-6 levels, L2H17 also inhibited their mRNA expression (Fig. 1, D and E) as well as the mRNA expression of other inflammatory markers such as IL-1β (Fig. 1F), IL-2 (Fig. 1G), IL-12 (Fig. 1H), and IFN-γ (Fig. 1I); all in a dose-dependent manner. We then determined the anti-inflammatory effects of L2H17 in PA-stimulated renal tubular epithelial SV40 cells. The results shown in Fig. 2 indicated that L2H17 dose-dependently inhibited L2H17-induced TNF-α and IL-6 releases at both the protein (Fig. 2, A and B) and mRNA (Fig. 2, C and D) levels. It is also observed that L2H17 treatment significantly reduced the expression of the cell adhesion molecules ICAM-1 and VCAM-1 (Fig. 2, E and F), which are responsive to the macrophage infiltration in chronic kidney diseases, in PA-stimulated SV40 cells.

Fig. 2.
Fig. 2.

L2H17 inhibited the PA-induced inflammatory response in SV40 cells. SV40 cells were pretreated with L2H17 at various doses (2.5, 5, or 10 μM), curcumin (10 μM), or vehicle [dimethylsulfoxide (DMSO) 3 μl] for 1 hour prior to stimulation with PA (100 μM) for different times. For 24-hour stimulation, enzyme-linked immunosorbent assay was used to detect the protein levels of TNF-α and IL-6 within the medium (A and B). For 6-hour treatment, total mRNA was extracted from the cell using TRIzol, and the mRNA levels of TNF-α (C), IL-6 (D), ICAM-1 (E), and VCAM-1 (F) were detected by real-time qPCR analysis. Curcumin at 10 μM was used as a comparison control. Bars represent the mean ± S.E.M. of four independent experiments performed in duplicate (*P < 0.05, **P < 0.01, and ***P < 0.001 versus the PA group).

L2H17 Suppressed PA-Induced Activation of NF-κB Signaling in MPMs.

In our previous studies, we demonstrated that chalones inhibited the expression of TNF-α via inactivation of NF-κB signaling in LPS-stimulated RAW 264.7 cells (Wu et al., 2011). Therefore, we hypothesized that L2H17 would exhibit a similar effect in PA-induced MPMs. Our results in Fig. 3 show that L2H17 dose-dependently inhibits the PA-induced activation of the NF-κB signaling pathway. IκB-α is a key component of the NF-κB pathway whose phosphorylation and degradation activates the NF-κB pathway. Western blot analysis showed that PA treatment increased phosphorylation and degradation of IκB-α (Fig. 3, A and B) as well as translocation of p65 from the cytoplasm to the nucleus (Fig. 3, C and D). In contrast, L2H17 dose-dependently decreased the phosphorylation and degradation of IκB-α (Fig. 3, A and B), inhibited the separation of IκB-α from the NF-κB p65 subunit, and reduced the release of p65 and its subsequent translocation from the cytoplasm to the nuclei (Fig. 3, C and D). We further evaluated the effects of L2H17 on the translocation of p65 through the use of immunofluorescence staining. As shown in Fig. 3E, the translocation of p65 from the cytoplasm to the nuclei was induced by PA and decreased following treatment with L2H17. EMSA also revealed that while PA lead to the increased ability of p65 to bind to the DNA, L2H17 significantly reduced the ability of p65 to bind with DNA (Fig. 3F). Curcumin was used as a comparison control. Taken together, these results suggest that L2H17 exhibits its anti-inflammatory activity through the suppression of the NF-κB signaling pathway.

Fig. 3.
Fig. 3.

L2H17 inhibited the PA-induced activation of NF-κB. MPMs were pretreated with L2H17 (2.5, 5, or 10 μM), curcumin (10 μM), or vehicle [dimethylsulfoxide (DMSO) 3 μl] for 1 hour prior to stimulation with PA (100 μM) for different times. For 30-minute stimulation, total proteins were extracted from the cell lysate and the level of phosphorylation and degradation of IκB was examined by Western blot with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (A and B). For 1-hour stimulation, the cytosolic and nuclear proteins were extracted, and the p65 levels were detected by Western blot using GAPDH and Lamin B as the loading controls (C and D); the translocation of p65 from the cytoplasm to the nucleus was detected by immunofluorescence (E); or the nuclear protein was isolated and mixed with a probe for NF-κB to detect the amount of p65 bound to DNA (EMSA method) (F). The column figures in (A–D) show the normalized optical density as a percentage of the PA group. Bars represent the mean ± S.E.M. of four independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001 versus the PA group).

L2H17 Suppressed the PA-Induced Activation of MAPK Signaling in MPMs.

It is well known that MAPK signaling plays an important role in the regulation of the synthesis of inflammation mediators and that PA can induce MAPK activity (Kaminska, 2005). Therefore, we investigated the effects of L2H17 on PA-induced MAPK signaling. As shown in Fig. 4A, PA can activate ERK, p38, and JNK, which comprise the three main MAPK signaling pathways in a time-dependent manner, whereas L2H17 was shown to inhibit those effects. Furthermore, Fig. 4B shows that L2H17 can also dose-dependently suppress the phosphorylation of ERK, p38 and JNK. This is more apparent when we examine the phosphorylated/unphosphorylated ratio for ERK, p38, and JNK (Fig. 4C) where we can see that while PA treatment resulted in an increased ratio for ERK, p38, and JNK. Pretreatment with L2H17 was able to significantly and dose-dependently reduce the ratio for all three. It is interesting to note that while curcumin had no effect on PA-induced p38 phosphorylation and activation, L2H17 inhibited PA-induced p38 activation. All of the above demonstrates that L2H17 had a significant effect in mitigating PA-induced inflammation through inhibition of both the NF-κB and MAPK signaling pathways.

Fig. 4.
Fig. 4.

L2H17 inhibited the PA-induced activation of MAPKs. (A) L2H17 inhibited PA-induced time-dependent phosphorylation of MAPKs. MPMs were pretreated with L2H17 (10 μM), curcumin (10 μM), or vehicle [dimethylsulfoxide (DMSO) 3 μl] for 1 hour and then stimulated with PA (100 μM) also for different time periods (0, 10, 20, 30, or 60 minutes). Total proteins were extracted from the cell lysate to detect for phosphorylation of MAPKs, such as ERK, p38, and JNK. (B and C) L2H17 inhibited PA-induced phosphorylation of MAPKs in a dose-dependent way. MPMs were pretreated with L2H17 (at 2.5, 5, or 10 μM), curcumin (10 μM), or vehicle (DMSO 3 μl) for 1 hour and then stimulated with PA (100 μM) for 30 minutes. Total proteins were extracted from the cell lysate to detect for phosphorylation of MAPKs. The bar graphs show the normalized optical density as a percentage of the PA group (C). Bars represent the mean ± S.E.M. of four independent experiments (*P < 0.05 and **P < 0.01 versus the PA group).

L2H17 Administration Did Not Affect Body Weight and Serum TCH and TG Levels in HFD-Fed Mice, but Had a Minor Effect on Serum LDL and HDL Levels.

To explore whether L2H17 had a similar effect in vivo, we set up an HFD-induced obesity mouse model and treated the mice with either L2H17 (20 mg/kg), curcumin (50 mg/kg), or vehicle control. As shown in Fig. 5, A–C the body weight, serum TCH levels, and serum TG levels were increased in the HFD-fed group when compared with the control group. Furthermore with regards to the body weight, TCH levels, and TG levels, there were no significant differences between the HFD-fed group and both the curcumin- and L2H17-treated groups. However with L2H17 treatment we noticed that there were significantly reduced LDL levels and elevated HDL levels (Fig. 5, D and E).

Fig. 5.
Fig. 5.

Administration of L2H17 had no effect on the body weight and serum TCH and TG levels, but had a minor effect on serum LDL and HDL levels. L2H17 did not affect body weight (A) and serum TCH (B) and TG (C) levels in HFD-fed mice. Body weight was measured at the time of death, and the blood was collected for serum analysis for TCH and TG. L2H17 decreased serum LDL (D) and increased serum HDL (E) as detected by their respective kits. Data are mean ± S.E.M. (n = 8 in each group; *P < 0.05 versus the HFD group).

L2H17 Administration Can Improve the Pathologic Lesions in the Kidneys of HFD-Fed Mice.

We then examined pathologic changes within the kidney tissues as well as in the key indicators of renal function, such as albumin (ALB) and creatinine (Cr). For our obesity model mice were fed a high fat diet for 8 weeks prior to treatment with L2H17 (20 mg/kg), curcumin (50 mg/kg), or vehicle control for 8 weeks. As shown in Fig. 6, HFD-fed mice exhibited elevated levels of ALB (Fig. 6A), which has been consistently observed in obesity-related kidney disease (Shen et al., 2010), and Cr (Fig. 6B) as well as an increased kidney/body weight ratio (Fig. 6C). However, L2H17 treatment drastically inhibited these parameters and was more effective than curcumin. Furthermore, we used H&E staining to observe the overall shape of the kidney. As we can see in Fig. 6D, L2H17 can significantly improved glomerular shrinking and prevented tubular necrosis. Further staining with Masson for type IV collagen and Sirius Red for fibrosis all indicated that L2H17 can improved the histologic changes of the kidneys of obese mice. Similar antifibrosis results were observed in the mRNA level. As shown in Fig. 6, E–H, a real-time qPCR assay revealed that L2H17 treatment significantly decreased HFD-induced overexpression of four fibrosis markers in the mouse kidney.

Fig. 6.
Fig. 6.

Administration of L2H17 improved kidney pathology and histology in HFD-fed mice. L2H17 lowered serum albumin levels (A), serum creatinine levels (B), and the kidney/body weight ratio (C) of HFD-fed mice. Body weight, kidney weight, and serum of mice were collected and analyzed at the time of death. (D) L2H17 significantly improved histologic abnormalities in the kidneys of HFD-fed mice. Displayed are representative figures of histologic abnormalities in the renal tissues of HFD-fed mice (400×). H&E staining was used for analysis of histologic abnormalities. Masson staining (blue) and Sirius red staining (red) were used for the detection of fibrosis in the kidney section. L2H17 administration significantly decreased HFD-induced expression of fibrosis markers, including vimentin (E), α–smooth muscle actin (SMA) (F), tumor growth factor (TGF) β (F), and collagen I (H) in the mRNA level. Data are mean ± S.E.M. (n = 8 in each group; *P < 0.05, **P < 0.01, and ***P < 0.001 versus HFD group).

L2H17 Administration Inhibited the Inflammation in the Kidney Tissues of HFD-Fed Mice.

We also wanted to examine if L2H17 had a similar anti-inflammatory effect in vivo in HFD-fed mice. As shown in Fig. 7A, anti–TNF-α immunofluorescence staining revealed that the kidneys of HFD-fed mice had increased TNF-α levels, which were significantly reduced with L2H17 treatment. When we further examined the mRNA expression of TNF-α (Fig. 7B) and other proinflammatory cytokines including IL-6 (Fig. 7C), IFN-γ (Fig. 7D), IL-1β (Fig. 7E), and anti-inflammatory cytokines such as IL-4 (Fig. 7F) and IL-10 (Fig. 7G), by real-time qPCR assay, it was observed that although HFD led to increased proinflammatory cytokines and decreased anti-inflammatory ones, L2H17 treatment reversed these HFD-induced changes with a higher ability than curcumin treatment. To confirm if these observed anti-inflammatory effects were due to suppression of the MAPK and NF-κB signaling pathways, we measured the levels of phosphorylated and unphosphorylated ERK, p38, JNK, and IκB-α. As expected and shown in Fig. 7H, the phosphorylation level of key components of the MAPK pathway, ERK, p38, and JNK were markedly increased in the HFD-fed group but reduced with the treatment with curcumin or L2H17. Furthermore, while HFD induced the phosphorylation of IκB-α and degradation of IκB-α, L2H17 treatment reversed HFD-induced degradation of IκB-α (Fig. 7H). These results again suggest that L2H17’s observed anti-inflammatory effects are due to the suppression of the MAPK and NF-κB signaling pathways.

Fig. 7.
Fig. 7.

L2H17 treatment attenuated the HFD-induced inflammatory response in the kidneys of HFD-fed mice. (A) L2H17 reduced the TNF-α expression in the kidneys of HFD-fed mice. The kidney tissues were stained for TNF-α (red), as described in Materials and Methods. L2H17 administration reduced the indicated gene expression in the kidneys of HFD-fed mice. Kidney tissues for mice in each group were collected for the mRNA detection of inflammatory markers, and subsequent gene analysis was done using real-time qPCR analysis for TNF-α (B), IL-6 (C), IFN-γ (D), IL-1β (E), IL-4 (F), and IL-10 (G) and normalized by β-actin (data are mean ± S.E.M., n = 8 in each group; *P < 0.05 and **P < 0.01 versus HFD group). (H) L2H17 significantly suppressed the activation of MAPKs and NF-κB. Tissues were homogenized for Western blot to analyze the phosphorylation and degradation of IκB and phosphorylation of MAPKs with ERK, p38, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading controls, respectively (for each group, three tissue samples from different mice were randomly selected and analyzed).

L2H17 Administration Reduced the Macrophage Infiltration and Expression of Adhesion Molecules in the Kidney Tissue of HFD-Fed Mice.

As macrophages in the tissue play an important role in the progression of renal damage and are one of the most critical contributors to end-stage diabetic nephropathy, we wanted to investigate if L2H17 could mitigate macrophage infiltration and expression of adhesion molecules in HFD-fed mice. As evidenced in Fig. 8A, staining for CD68 reveals that L2H17 attenuated against HFD-induced macrophage infiltration. Furthermore, our results reveal that L2H17 also decreased both the mRNA (Fig. 8, B and C) and protein expression of the adhesion molecules ICAM-1 and VCAM-1, respectively (Fig. 8D). We also observed the profile of chemokine and the chemokine receptor in the kidneys of L2H17-treated HFD mice. Figure 8, E and F, showed that treatment with L2H17 or curcumin significantly decreased the HFD-induced mRNA overexpression of MCP1 and CCR2.

Fig. 8.
Fig. 8.

Administration of L2H17 inhibited the expression of adhesion molecules and reduced macrophage infiltration. After the mice were sacrificed, the kidney tissues in each group were collected for further analysis. (A) L2H17 attenuated macrophage infiltration in the kidney tissues of HFD-fed mice. CD68 staining was used for the detection of macrophage infiltration (400×). (B–F) L2H17 treatment decreased expression of the adhesion molecules and chemokines. Kidney tissues were homogenized for real-time qPCR assay for the mRNA levels of ICAM-1 (B), VCAM-1 (C), MCP-1 (E), and CCR2 (F) or for Western blot analysis for the protein levels of ICAM-1 and VCAM-1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (D), respectively. Data are mean ± S.E.M. (n = 8 in each group; *P < 0.05, **P < 0.01, and ***P < 0.001 versus HFD group).

Discussion

Due to the rise in obesity rates worldwide, we have seen a similar increase in the cases of ORG. While it has been long-established that obesity is a risk factor for the development of cardiovascular disease and diabetes mellitus, more recent research points to obesity as an important risk factor for the development of chronic kidney disease. A large-scale study conducted using 6818 liver biopsies from 1986 to 2000 showed that over the course of 15 years the prevalence of ORG rose from 0.2% in 1986–1990 to 2.0% in 1996–2000 (Kambham et al., 2001). However, despite significant increases in the prevalence of ORG, therapeutic options are still very limited and, as a result, we are also seeing an increase in the cases of end-stage renal disease. Subsequently, the need for finding new therapeutic options for the treatment of ORG has never been greater.

Obesity is now considered a state of chronic low-grade inflammation. Increased inflammation in the adipose and kidney tissues promotes the progression of kidney damage in obesity (Wei et al., 2004; Hunley et al., 2010; Gregor and Hotamisligil, 2011; Manabe, 2011). Furthermore, hyperlipidemia itself induces inflammation and results in the disruption of cell function and pathologic changes in renal glomeruli (Kershaw and Flier, 2004; Hunley et al., 2010). Therefore, the reduction of inflammatory cytokines through anti-inflammatory therapy might lead to the prevention and/or treatment of ORG. Our group has shown that the inflammatory response is involved in diabetes-induced renal damage, and others have already demonstrated that compounds with anti-inflammatory properties, such as thiazolidinedione and curcumin, can ameliorate diabetes-induced renal injury (Ohga et al., 2007; Pan et al., 2013b).

Chalcones are a class of naturally occurring open-chain flavonoids that have gained increased interest for their many therapeutic properties, including their anti-inflammatory activity. These observations prompted us to synthesize a number of chalcone derivatives and screen them for anti-inflammatory activity (Wu et al., 2011). When we found that one of the derivatives, L2H17, had significant anti-inflammatory properties in LPS-induced inflammation in macrophages, we questioned if these anti-inflammatory effects could extend to treating obesity-related disorders, such as ORG. From the results of this study, we have confirmed L2H17’s anti-inflammatory activity both in PA-treated MPMs and HFD-fed mice and that L2H17 significantly attenuated obesity-induced renal injury through inhibition of the NF-κB/MAPK–dependent inflammatory cytokine production.

The NF-κB pathway plays an important role in the regulation of the inflammatory response, with downstream targets that include proinflammatory cytokines, chemokines, cell adhesion molecules, regulators of cell apoptosis, and stress response genes (Oeckinghaus and Ghosh, 2009). When inactive, NF-κB dimers are sequestered in the cytoplasm by a family of inhibitors known as IκBs. Of the IκBs, IκB-α is the major and most studied one. The pathway is activated when IκB-α is phosphorylated, which results in its dissociation from the NF-κB cytoplasmic complex. Once activated, the subunit of NF-κB, p65, migrates into the nucleus where it is then able to activate the expression of its downstream targets (Huxford et al., 1998; Jacobs and Harrison, 1998; Gilmore, 2006; Adli et al., 2010). It has been demonstrated before that high glucose increases NF-κB transcription activity through the increased phosphorylation of IκB (Lee et al., 2010). In MPMs we observed that PA activated the NF-κB pathway, as evidenced by decreased levels of unphosphorylated IκB and cytoplasmic p65, and increased levels of phosphorylated IκB and nuclear p65 (Fig. 3, A–D). Immunofluorescence staining revealed the translocation of p65 from the cytoplasm to the nucleus (Fig. 3E). EMSA showed the increased ability of nuclear p65 to bind to the DNA (Fig. 3F). Furthermore, when we examined the expression of proinflammatory cytokines, we found that PA treatment resulted in increased protein and mRNA levels of key proinflammatory cytokines (Fig. 1, D–I). However, pretreatment with L2H17 was able to reverse all of the above effects in a dose-dependent manner, increased levels of unphosphorylated IκB, decreased translocation of p65 from the cytoplasm to the nucleus, decreased p65’s ability to bind to the DNA (Fig. 3), and ultimately decreased protein and mRNA expression of key proinflammatory cytokines (Fig. 1, B–I). Similar results were also observed in the kidney tissues of HFD-fed mice (Fig. 7). Furthermore, in both in vitro and in vivo studies, L2H17 surpassed curcumin in efficacy in most cases at the dose of 2.5 μM and in all cases at 5 μM. These results suggest that L2H17 is able to effectively mediate and inhibit the inflammatory response through the inactivation of the NF-κB signaling pathway.

The MAPK pathway has also been shown to play an important role in obesity and its activation has been implicated in obesity-related complications such as ORG (Yan et al., 2013). JNK, p38, and ERK, the three main subfamilies of MAPKs, have all been reported to be activated in response to inflammatory and stress-inducing stimuli. Previous studies have shown that all three were activated in high glucose-induced NRK-52E cells (Bost et al., 2005; Kaminska, 2005; Pan et al., 2013a). As expected, PA-induced MPMs also exhibited increased phosphorylation of ERK, JNK, and p38 as evidenced by increased p-ERK/ERK, p-p38/p38 and p-JNK/JNK ratios (Fig. 4C), all in a dose-dependent manner. While curcumin had no observed effect on the phosphorylation of p38, L2H17, even at 2.5 μM, had a significant effect on reducing PA-induced increases in the phosphorylation of p38. Similar results were observed in HFD-fed mice (Fig. 7H). In both MPMs and HFD-fed mice, L2H17 treatment inhibited PA/HFD-induced MAPK activation, decreased phosphorylation of ERK, p38, and JNK.

In the kidney tissues of HFD-fed mice, we observed how HFD induced pathologic and histologic changes. We noticed significant increases in serum ALB levels (Fig. 6A)-a hallmark of obesity-induced kidney injury-elevated serum Cr levels (Fig. 6B), and an increased kidney weight/body weight ratio (Fig. 6C). Further H&E staining revealed significant structural abnormalities. Masson/Sirius Red staining and a real-time qPCR assay revealed increased renal fibrosis and collagen accumulation (Fig. 6, D–H). The administration of L2H17 improved kidney pathology and histology in HFD-fed mice, reduced ALB and Cr levels as well as decreased the kidney weight/body weight ratio. Staining revealed improvements in histologic abnormalities, glycogen accumulation, and fibrosis. These results support L2H17’s ability to protect against HFD-induced renal injury.

Obesity-related macrophage infiltration of adipose tissue is believed to be key in inflammation and insulin resistance, and experimental studies have shown that inhibiting proinflammatory macrophages successfully reduces renal injury (Nguyen et al., 2006). Cell adhesion molecules, such as VCAM-1 and ICAM-1, play an important role in the recruitment of macrophages. Gene expression and protein levels of VCAM-1 and ICAM-1 have been identified in the early stages of diabetic renal injury both in human and animal models (Nguyen et al., 2006; Fang et al., 2015). It has been reported that NF-κB regulates the gene expression of adhesion molecules in renal cells, and recent reports have also implicated p38 and JNK pathways as well (Navarro-Gonzalez et al., 2011; Pan et al., 2013a). In this study, we observed-through CD68 staining-an increase in macrophage infiltration in the kidney tissues of HFD-fed mice that was attenuated with treatment of L2H17 (Fig. 8A). Furthermore, L2H17 also mitigated HFD-induced increases in cellular adhesion molecules such as ICAM-1 and VCAM-1, chemokine MCP-1, and chemokine receptor CCR2 in the renal tissues of HFD-fed mice (Fig. 8, B–F). These results suggest that L2H17 can protect against renal injury through the inhibition of macrophage infiltration.

The results of this study indicate that L2H17 is an effective therapeutic agent for the treatment of obesity-related glomerulopathy. Although in vivo, we can see that L2H17 treatment did not induce any significant changes in body weight, total glucose, or total cholesterol levels; there was an observable reduction in LDL levels and an increase in HDL levels (Fig. 5), suggesting that the mechanism behind L2H17’s effects may be multifaceted. Moreover, we can prominently see that L2H17 had a marked effect on PA/HFD-induced inflammation. These results indicate that L2H17’s protective effects against renal injury are most likely attributed to its anti-inflammatory properties, which we believe are due to its inhibition of the NF-κB and MAPK pathways. Furthermore, in most cases, L2H17 exhibited greater pharmacological activity at lower concentrations than curcumin. However, it still remains unclear whether the beneficial effects of L2H17 are NF-κB/MAPK dependent, and while we have been able to observe L2H17’s inhibition of both pathways, its molecular target(s) remain unknown. Therefore, we believe that further studies are necessary to elucidate the underlying molecular mechanisms and targets of L2H17. Regardless, the findings reported in this study demonstrate that targeting the NF-κB/MAPK pathways may be an important therapeutic target and suggests that L2H17 is a promising anti-inflammatory agent in the treatment of obesity-related complications, such as ORG.

Authorship Contributions

Participated in research design: Liang, Tong, Pan.

Conducted experiments: Fang, Deng, L. Wang, Zhang.

Contributed new reagents or analytic tools: Yin.

Performed data analysis: Weng.

Wrote or contributed to the writing of the manuscript: Liang, J. Wang.

Footnotes

    • Received June 17, 2015.
    • Accepted September 8, 2015.

  • Q.F. and L.D. contributed equally to this work.

  • This study was supported by the Natural Science Foundation of China [Grants 81300678, 81200572, 81302821, and 81371028], Zhejiang Provincial Natural Science Funding [Grants LQ14H310003 and LQ13H310002], and High-Level Innovative Talent Funding from the Zhejiang Department of Health [Grant 2010-017].

  • dx.doi.org/10.1124/jpet.115.226860.

Abbreviations

ALB
albumin
BSA
bovine serum albumin
CCR2
C-C motif chemokine receptor 2
CKD
chronic kidney disease
Cr
creatinine
EMSA
electrophoretic mobility shift assay
ERK
extracellular signal-related kinase
FFA
free fatty acid
HDL
high-density lipoprotein
H&E
hematoxylin and eosin
HFD
high fat diet
HRP
horseradish peroxidase
IκB
inhibitor of nuclear factor κB
ICAM-1
intercellular cell adhesion molecule 1
IFN
interferon
IL
interleukin
JNK
c-Jun N-terminal kinase
LDL
low-density lipoprotein
LPS
lipopolysaccharide
L2H17
1-(3,4-dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one
MAPK
mitogen-activated protein kinase
MCP
monocyte chemotactic protein
MPM
mouse peritoneal macrophage
NF
nuclear factor
ORG
obesity-related glomerulopathy
PA
palmitic acid
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
qPCR
quantitative polymerase chain reaction
TCH
total cholesterol
TG
triglyceride
TNF
tumor necrosis factor
VCAM-1
vascular cell adhesion molecule 1

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Research ArticleInflammation, Immunopharmacology, and Asthma

Chalcone Prevents ORG via Anti-Inflammatory Action

Qilu Fang, Liancheng Deng, Lintao Wang, Yali Zhang, Qiaoyou Weng, Haimin Yin, Yong Pan, Chao Tong, Jingying Wang and Guang Liang
Journal of Pharmacology and Experimental Therapeutics November 1, 2015, 355 (2) 235-246; DOI: https://doi.org/10.1124/jpet.115.226860
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