Renoprotective Effect Of Isoliquiritigenin On Cisplatin-induced Acute Kidney Injury Through Inhibition Of FPR2 in Macrophage

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Tan Rui-Zhi et al

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Abstract

Acute kidney injury (AKI) is a serious complication in critically ill patients. Accumulating evidence indicated that macrophages play an important pro-inflammatory role in Acute Kidney Injury and isoliquiritigenin (ISL)can inhibit macrophagic inflammation, but its role in Acute Kidney Injury and the underlying mechanism is unknown. The present study aims to investigate the renoprotective effect of ISL on AKI and the role of Formylpeptide receptors 2 (FPR2) in this process. In this study, the cisplatin-induced AKI model and lipopolysaccharide-induced macrophage inflammatory model were employed to perform the in vivo and in vitro experiments. The results showed that ISL strongly relieved kidney injury and inhibited renal inflammation in vivo and suppress the macrophagic inflammatory response in vitro. Importantly, it was found that FPR2 was significantly upregulated compared to the control group in AKI and LPS-induced macrophage, whereas it was strongly suppressed by ISL. Interestingly, overexpression of FPR2 with transfection of pcDNA3.1-FPR2 effectively reversed the anti-inflammatory effect of ISL in macrophage, suggesting that FPR2 may be the potential target for ISL to prevent inflammation and improve kidney injury of Acute Kidney Injury. Taken together, these findings indicated that ISL improved cisplatin-induced kidney injury by inhibiting FPR2 involved macrophagic inflammation, which may provide a potential therapeutic option for AKI.

1. Introduction

Acute kidney injury (AKI) is a common severe type of kidney disease with high mortality.1 It is characterized by a rapid decline in renal function that occurs within 7 days or less. Recent publications reported that the incidence of AKI is as high as 5% of inpatients and 30% of severe patients, and AKI causes 2 million deaths worldwide each year, which has become a global health problem. Although the definition is ordinary and uniform, AKI can be caused by a variety of reasons, including nephrotoxic drug intake, ischemia-reperfusion injury (Ischemia-reperfusion injury, IRI), sepsis, cardiovascular surgery, hypertension, and diabetes.2,3 Old age, diabetes, and heart failure increase the risk of AKI, which usually leads to irreversible chronic kidney disease (CKD) and end-stage renal disease (ESRD) in some patients.4 Notably, studies have shown that almost all immune cells are involved in the immune inflammation of the kidney in AKI.5 Among them, macrophages have been shown to have an important role in promoting renal inflammation.6 Deleting macrophages improve AKI kidney injury and inflammation.7 Therefore, effective inhibition of macrophagic inflammation may be beneficial for improving the current therapeutic approaches for the management of AKI.

Isoliquiritigenin (ISL) is a flavonoid with a chalcone structure, which is rooted in the famous traditional Chinese herbal medicine.8 In recent years, it has attracted wide attention from scientists due to its antioxidant, anti-inflammation, anti-platelet aggregation, antibacterial, anti-viral, anti-tumor, hepatoprotective, neuroprotective, cardiac protection, and detoxification properties.9 ISL has been shown to inhibit cell proliferation, reduce ROS, and induce apoptosis, necrotic, and death of cancer cells to various degrees.10 Recent reports indicated that isoliquiritigenin can inhibit the production of IL-6 and TNF-a induced by the lipid A portion of LPS in RAW264.7 macrophages and attenuate the formation of LPS-TLR4/ MD-2 complex. In addition, ISL can induce HepG2 cell apoptosis through ROS-mediated MAPK, STAT3, and NF-kB signaling pathways.11 Experimental pieces of evidence also showed that ISL induces ROS-mediated intrinsic activation and activates the mitochondrial-cytochrome c-cysteine protease mechanism of PC-12 cell apoptosis. It is worth noting that ISL is often used as a dietary supplement for women.12 Recent studies have shown that ISL has a significant anti-inflammatory effect on AKI, but the specific mechanism of action is still unclear.

Previous research from our team found that Mincle, the macrophage-induced C-type lectin, is a transmembrane pattern recognition receptor involved in renal inflammation,13 through which, we screened formyl peptide receptors 2 (FPR2) that has a certain relationship with Mincle from the GEO database. However, its role in acute kidney disease has not been verified. FPR2 is a protein-coding gene that belongs to the G protein-coupled receptor family and contains 351 amino acid residues.14,15 They are 7- transmembrane G protein-coupled receptors that can mediate immune regulation, inflammatory factor secretion, cell chemotaxis, and cell adhesion, which are closely related to the occurrence and development of various diseases.16 Its homology with FPR1 and FPR3 is as high as 69% and 83%. FPR2 subcells are localized in the cell membrane and are multi-pass membrane proteins. They are mainly expressed in neutrophils, eosinophils, monocytes, epithelial cells, fibroblasts, glial cells, mesangial cells, etc., distributed in various organs such as the lung, spleen, and liver. Notably, FPR2 is abundantly expressed in the renal interstitium,17 where inflammatory cells reside. The main function of FPR2 is to combine certain specialized decomposition-promoting media (SPM) to mediate the activity of these metabolites in suppressing and eliminating inflammation. However, the role of FPR2 in AKI inflammation and whether it can be used as a target for a drug intervention is still unknown.

In this present study, we employed a nephrotoxic drug -cisplatin-induced mouse AKI model and LPS induced inflammatory cell model to explore the improvement effect of ISL on injury and inflflammation of kidneys in AKI, as well as the key role of FPR2 in renal inflammation and ISL treatment of AKI. This study will provide new directions for understanding the mechanism of inflammatory injury and treatment strategies for AKI.

2. Materials and methods

2.1. Drugs

Isoliquiritigenin (purity> 98%) is an isoflavone compound extracted from licorice root. Its molecular formula is C15H12O4. In this study, isoliquiritigenin was purchased from Chengdu Biopurify Phytochemicals Ltd. (BP0787), dissolved in 10% DMSO(ST038, Beyotime, China), and cisplatin-induced mice were intragastrically administered in a dose of 7.5 mg/kg and 30 mg/kg, respectively.

2.2. Isolation of BMDM

Male C57BL/6 mice with ages of 6e8 weeks were selected to isolate the BMDM cells. Carefully separate the hind leg bone from the femur and remove the attached muscles. Grind the bones in PBS with a mortar until the color of the bones changes from red to white. Subsequently, collect the supernatant and filter through a 70 strainer, followed by centrifuging at 1500 rpm for 5 min. The erythrocytes were broken in filtered liquid with 10 - PBS, which was then filtered on a 40 mm cell strainer. Incubate the obtained cells in a 10 cm culture dish and induce with 30% L929 supernatant for 7 days. The BMDM cells were stimulated with 100 ng/ml LPS to construct the inflammatory cell model.

2.3. Knock-down and overexpression of FPR2 in BMDM

FPR2 was down-regulated by transfection with Zeta LifeAdvanced Transfection Reagent (AD600050, Zeta life, USA) andFPR2 siRNA to BMDM (The sequences of FPR2 siRNA: Sense, 5’-GGAGAUGUGUAUUGUACAUTT-3’; Antisense, 5’-AUGUACAAUACACAUCUCCTT-3’. Sangon Biotech, China). Similarly, pcDNA3.1-FPR2plasmid (Constructed by Fenghui Biotech, China) was transfected into BMDM with ZETA to overexpress FPR2 in cells.

2.4. Animal experiments

All animal experiments were performed according to the guidelines from the Ethics Committee of Southwest MedicalUniversity. 32 male C57BL/6 mice were purchased from ChengduDaShuo Animal Co., Ltd, which were randomly divided into 4groups: sham group, model group (Cisplatin-induced AKI group), ISL low dose group (7.5 mg/kg, group L), and high dose group(30 mg/kg, group H). The model group was injected with 20 mg/kg cisplatin (P4394, Sigma-Aldrich, USA) on the first day, while the low dose group was given 7.5 mg/kg ISL and the high group was given30 mg/kg ISL for 3 days. After 3 days, serum and kidney were collected from the sacrificed mice.

2.5. Renal function test

After collecting the serum of each group of mice, strictly follow the instructions of kits to detect the levels of serum creatinine(C011-2-1, Jiancheng Biotech, China) and urea nitrogen (C013-2-1, Jiancheng Biotech, China).

2.6. Pathological staining

H&E staining: Section routinely dewaxing and rehydration, hematoxylin staining for 5 min, RO water rinse, 1% hydrochloric acid alcohol separation for 2 s, tap water rinse, 75% ethanol immersion for 2 s, 1% alcohol-soluble eosin for 30 s, tap water rinse,100% ethanol Dehydrate for 1 min, dry, xylene transparent for10 min, dry, seal with neutral balsam mounting medium(36313ES60, Yepsen, China).

PAS staining: The slices were placed in periodic acid solution at room temperature for 5e8 min, followed by rinsing twice with RO water. Then, put these slices into Schiff reagent for 10e20 min, rinsed with tap water for 10 min, stained in hematoxylin for1e2 min, dehydrated and transparent in 100% alcohol, dry, seal with neutral balsam mounting medium.

2.7. Immunoflfluorescence

Place frozen tissue sections in PBS, wash twice, fix with 10%formaldehyde for 10 min. The tissue on sections was blocked with 5%goat serum for 30 min, followed by incubating with primary mouse anti-mouse FPR2 (1:200, sc-57141, Santa Cruz, USA) and rat anti-mouse F4/80 antibodies (1:200, sc-52664, Santa Cruz, USA) at4 - C overnight. The next day, wash the sections twice with PBS, incubate with secondary antibody (1: 200) at room temperature for1 h, and stain the nucleus with DAPI. The images were captured byEVOS system.

2.8. Elisa

The concentration of IL-1b, IL-6, TNF-a in serum and cellular supernatant were analyzed using ELISA Kits (EMC001B, EMC004, EMC102A, Neobioscience, China) according to the kit instructions.

2.9. RealTime-PCR

Total RNA was extracted using an RNA extraction kit (Tiangen, China) following the manufacturer's instructions. After reversely transcribing the extracted RNA into cDNA, adding it to a 10 mL reaction system to perform the real-time PCR by Roche LightCycler 480 system and the reaction system is as follow: Eastep® qPCR MasterMix 2 -, 5 mL; cDNA template, 1 mL; upstream primer (10 mM), 0.5 mL; downstream primer (10 mM), 0.5 mL; Nuclease-free water, 3 mL. Primers used in this experiment were synthesized by Sangon (Shanghai, China), and the sequences of all primers are listed in Table 1.

2.10. Western blot

RIPA lysis buffer was used to extract protein from kidney tissues and BMDM cells. After resolving protein in 10% SDS-PAGEgel and transferring to PVDF membrane, blocking the membrane in 5% BSA for 1 h and washed with TBST 3 times, followed by incubating the membrane with primary antibodies at 4 - C overnight (prepared with 5% BSA, 1: 1000). Then, the membranes were incubated with the secondary antibody (Peroxidase-Conjugated Goat anti-Mouse IgG (ZB-2305, 1:3000), Goat anti-Rabbit IgG (ZB-2301, 1:3000)) at room temperature for1 h. The images were captured by a gel imaging system (BIORAD, USA). The gray intensity of the bands was calculated by Image J software.

2.11. Statistical analysis

Results were shown as mean ± standard deviation. Data analysis was performed with a one-way ANOVA test followed by multiple-comparison with LSD test by using SPSS software 21.0. P < 0.05 was considered statistically signifificant. Each group of experiments duplicates 3 times.

3. Results

3.1. Isoliquiritigenin improved cisplatin-induced kidney injury and inhibited renal inflammation

To investigate the renoprotective and anti-inflammatory effects of isoliquiritigenin in AKI, we detected the levels of serum Cre and BUN of each group, as well as the expression and secretion of in- inflammatory cytokine by real-time PCR and ELISA. In addition, the renal pathology was measured by H&E and PAS staining. The results showed that compared with the normal group, the levels of serum Cre and BUN were increased in the AKI model, while treatment with 7.5 mg/kg or 30 mg/kg of ISL significantly reduced the levels of serum Cre and BUN, among them, 30 mg/kg dose has the best therapeutic effect (Fig. 1A and B). Notably, 30 mg/kg of ISL can improve renal tubular damage according to the evaluated tubular necrosis percent (Fig. 1C). The ELISA and PCR results showed that the secretion of IL-1b, IL-6, and TNF-a has downregulated after ISL (7.5 mg/kg and 30 mg/kg) intervention, and the mRNA expression of IL-1b, IL-6, and TNF-a were also remarkably reduced by administration of 30 mg/kg of ISL (Fig. 1DeI). On the other hand, the results of H&E and PAS staining demonstrated that the kidney in the AKI group presented severely renal tubular damage, renal epithelium shed, mesangial cells and mesangial interstitial hyperplasia, and inflammatory cells infiltration, surprisingly, 30 mg/kg of ISL can effectively reverse the injury of kidney in AKI (Fig. 1J). All the results revealed that administration of 7.5 mg/kg and 30 mg/kg ISL can both improve cisplatin-induced kidney injury and inhibit renal inflammation, in which, 30 mg/kg ISL has the best effect.

3.2. Isoliquiritigenin reduced the expression of FPR2 in macrophages of AKI mice

A lot of previous reports indicated that FPR2 plays a vital role in immune response and inflammation-related diseases. However, the potential effect of FPR2 in the kidney of AKI is undefined. In this study, we performed immunofluorescence to co-locate the FPR2 and macrophages (F4/80, a maker of macrophages). The results showed that macrophages (green spots) were voluminously infiltrated in the kidney of AKI mice, meanwhile, FPR2 (red spots) was co-located with F4/80, illustrating that FPR2 was mainly expressed on macrophage and the level of FPR2 was significantly increased in the kidney of AKI mice (Fig. 2A). We further detected the expression of FPR2 mRNA and inflammation-related proteins by real-time PCR and western blotting in the kidney, and the results demonstrated that 30 mg/kg of ISL can effectively down-regulate the mRNA and protein levels of FPR2 in the kidney of AKI mice compared with 7.5 mg/kg of ISL group (Fig. 2B, G, H). Importantly, the western blotting results showed that 30 mg/kg of ISL also inhibited the activation of p-65 and down-regulated the protein levels of TNF-a, IL-6, and IL-1b in the kidney of AKI mice (Fig. 2CeF). All the above data suggested that ISL relieved renal inflammation of AKI mice may relate to reducing the expression of FPR2 in macrophages.

3.3. Isoliquiritigenin suppressed the expression and secretion of FPR2and inflammatory cytokines in LPS-induced BMDM cells

To further detect the effects of ISL on LPS-inducedBMDM cells, we examined the mRNA level of FPR2, expression, and secretion of inflammatory cytokines in LPS-induced BMDM cells after ISL administration by real-time PCR and ELISA. The results demonstrated that ISL decreased the mRNA level of FPR2, IL-1b, IL-6, and TNF-a (Fig. 3AeD), as well as the secretion of IL-1b, IL-6, and TNF-a in cellular supernatant in LPS-induced BMDM cells (Fig. 3EeG). The above results suggested that treatment of ISL with the concentration of 40 mM strongly inhibited inflammation in LPS-induced BMDM cells.

3.4. Isoliquiritigenin downregulated the protein levels of FPR2 and inflammatory cytokines in LPS-induced BMDM cells

We also detected the protein levels of FPR2 and inflammatory cytokines in LPS-induced BMDM cells after ISL administration by western blotting. The data showed that compared with the control group, the protein levels of FPR2, p-p65, IL-6, and TNF-a were significantly increased in LPS-induced BMDM cells, and strongly reduced with 40 mM ISL administration (Fig. 4AeE). These results further illustrated that ISL can suppress the activation of NF-kB and down-regulate protein levels of inflammatory cytokines in LPSinduced BMDM cells.

3.5. Inhibition of FPR2 suppressed LPS-induced inflammation in BMDM cells

To explore the role of FPR2 in macrophage inflammation, we used siRNA to knock-down FPR2 in BMDM cells. The results showed that siRNA transfection can significantly inhibit the expression of FPR2 in LPS-induced macrophage (Fig. 5A), and effectively reduce the expression of IL-1b and TNF-a (Fig. 5B and C). In addition, western blot results proved that after siRNA transfection, the protein level of FPR2 was significantly inhibited, regardless of whether it was stimulated by LPS (Fig. 5D and E). Notably, knocking down of FPR2 significantly reduced the protein levels of inflammatory cytokines and inhibited the activity of NF-kB signaling (Fig. 5FeK), indicating that FPR2 plays an important role in promoting the inflammatory response in macrophages. Also, it may be the key target of ISL to inhibit macrophage inflammation.

3.6. Overexpression of FPR2 counteracted the anti-inflammatory effect of isoliquiritigenin in LPS-induced BMDM cells

To determine that ISL suppresses inflammation in AKI through inhibiting FPR2 in macrophage, we used FPR2 overexpression plasmid to up-regulate the expression of FPR2 in ISL treated inflammatory BMDM cells. As shown in Fig. 6, the protein and mRNA level of FPR2 were significantly increased in the pcDNA3.1- FPR2 transfected group (Fig. 6A, B, G), and overexpression of FPR2 effectively abolished the inhibitory effect of 40 mM ISL on the activation of p65 and the protein levels of IL-1b, IL-6 and TNF-a in LPS-induced BMDM cells (Fig. 6CeF). Similarly, up-regulated of FPR2 removed the suppressive effect of 40 mM ISL on expression and secretion of inflammatory cytokines in LPS-induced BMDM cells, such as IL-1b, IL-6, and TNF-a (Fig. 6HeM), suggesting that ISL reduced inflammation in LPS-induced BMDM cells by inhibiting FPR2.

4. Discussion

Acute kidney injury is one of the common clinical illnesses. It usually leads to the impaired immune function of patients in a short period. In cases of trauma blood loss, organ damage, shock, the kidney is the most susceptible organ and is prone to develop into an acute injury.18e20 Numerous studies have confirmed that inflammation plays a key role in the development of AKI, which has been considered as a potential intervention target for AKI treatment. In recent years, Chinese medicines have made tremendous progress in the intervention of inflammation, including curcumin, quercetin, naringenin, and other monomers. Iso-liquiritigenin is a flavonoid compound, which has many biological activities, such as anti-inflammatory, anti-free radical, anti-tumor, and other effects.21 Previous studies found that ISL inhibited apoptosis, inflammation, and extracellular matrix production in angiotensin II-induced HK-2 cells.22 Studies from Wu Yangping showed that ISL can down-regulate IL-6 and IL-8 by inhibiting the activity of NF-kB in the psoriasis model, suggesting ISL may be a candidate for the treatment of psoriasis and other autoimmunity inflammatory diseases.23 On the other hand, a recent study demonstrated that ISL could alleviate septic AKI by suppressing NF-kB p65 translocation and inhibiting inflammatory responses.24 In this study, we found that ISL can effectively improve the renal function in cisplatin-induced AKI mice, reduce kidney damage, and inhibit renal inflammation. However, the precise mechanism of ISL inhibiting inflammation is unclear.

FPR2 belongs to the family of chemotactic receptors and is a G protein-coupled receptor of 7 times transmembrane. Previous studies have found that FPR2 is involved in a variety of physiological and pathological processes, including defense responses, inflammation, neurodegenerative diseases, tumors, disorders related to glucose and lipid metabolism.25,26 FPR2 can be upregulated by lipopolysaccharides, various inflammatory factors, and prostaglandin E2 in the inflammatory process which may play an anti-inflammatory and pro-inflammatory role.27,28 The different effects of FPR2 on inflammation depend on the environment and ligands that activate different signaling pathways.29 In this study, we found that FPR2 is mainly expressed in macrophages in the renal interstitium, and is highly expressed in the kidney of cisplatin-induced AKI mice, suggesting that FPR2 in macrophages may be involved in the inflammatory response of macrophages in AKI kidney. Increasing pieces of evidence have confirmed that a large number of macrophages infiltrated into the kidney within a few hours after the occurrence of AKI, and aggravated kidney damage by responding to inflammation.30e33 Therefore, down-regulating the inflammation of infiltrated macrophage in AKI kidney may be an important direction to improve AKI, and whether FPR2 is involved in this process is unknown.

Our experimental results showed that treatment with low- and high-dose ISL in cisplatin-induced AKI mice, not only can improve renal structure and function, but also reduce the level of inflammatory cytokines. In addition, ISL can effectively down-regulate the protein level of FPR2 and the activity of p65. Since FPR2 is mainly expressed in macrophages, it indicates that ISL may improve kidney inflflammation by inhibiting FPR2 in macrophages. Because of the important role of the infiltrated macrophage in renal inflflammation, we then extracted primary bone marrow-derived macrophages for in vitro detection of the improvement effect of ISL on macrophages inflammation and the potential mechanism. Our results showed that ISL can significantly inhibit LPS-induced macrophage inflammation and down-regulate the expression and secretion of in-inflammatory factors IL-1b, IL-6, and TNF-a. At the same time, the ISL can also reduce the protein level of inflammatory molecules in macrophage and inhibits the mRNA expression and protein levels ofFPR2 stimulated by LPS. To verify the key role of FPR2 inhibition of inflflammation with ISL, we used the pcDNA3.1-FPR2plasmid to overexpress FPR2 and found that in the inflammatory macrophage treated with ISL, overexpression of FPR2 neutralize the inhibitory effect of ISL on inflflammation, indicating that ISL down-regulated macrophage inflflammation is mainly through the inhibition of FPR2. Notably, we used the cisplatin-induced AKI model in vivo and selected the LPS-induced inflammatory cell model in vitro. Although the induction reagents of the two models are not consistent, these two methods are currently recognized to construct inflflammation models in vivo and in vitro, respectively. Given that this research focuses on inflammatory responses, it is feasible to use these two methods in one study.

These data proved that ISL can effectively reduce the expression of inflammatory factors in vivo and in vitro, and may be achieved by inhibiting the expression of FPR2. ISL may thus serve as a potential therapeutic drug for AKI.

Acknowledgments

This work was supported by the Luzhou - Southwest MedicalUniversity Joint Project (2017LZXNYD-P01 and 2018LZXNYD-PT03), the Southwest Medical University - Affiliated Traditional MedicineHospital Joint Project (2018XYLH-034), the Construction Project of Sichuan Provincial Key Laboratory of Medicine (2018#53), the Luzhou science and technology project (2020-JYJ-56) and the Luzhou Municipal - Southwest Medical University Joint SpecialGrant for the Introduction of High-level Talents (Lan Hui-Yao Team).

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