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Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats
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Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Available online at
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Original article
Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats Long Hongyana , Wu Sulingb,* , Zhu Weinaa , Zhang Yajiea , Ruan Jiea a b
Central Laboratory, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing 210001, China Department of Rheumatology, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing 210001, China
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 25 October 2016 Accepted 1 November 2016 Keywords: Liquiritigenin Hyperuricemia AQP4 NLRP3 Inflammation
A B S T R A C T
The aim is to investigate the anti-hyperuricemic and renal protective effects of liquiritigenin in potassium oxonate-induced hyperuricemic rats. Hyperuricemia in rats was induced were induced with potassium oxonate (250 mg/kg) intragastrically for 7 days, and liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/ kg) were daily administrated to the rats orally 1 h after the potassium oxonate exposure. Liquiritigenin significantly reversed the elevated productions of uric acid in serum and urine and pro-inflammation cytokines in serum and kidney, which shown that liquiritigenin has renal protective effects. Histological study shows that liquiritigenin inhibited severe necrosis and inflammatory cell infiltration in potassium oxonate-treated rats. Furthermore, liquiritigenin mediated the activities of aquaporins 4 (AQP4), and regulated the activation of NF-kB p65 and the degradation of IkBa. Finally, significant increases of nodlike receptor protein 3 (NLRP3) inflammasome, apoptosis-associated speck-like protein adaptor (ASC) adaptor and cleaved caspased-1 were restored by liquiritigenin. Therefore, liquiritigenin might improve renal inflammation by suppressing renal AQP4/NF-kB/IkBa and NLRP3 inflammasome activation in hyperuricemic rats. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Hyperuricemia, a high-prevalence metabolic disease, is usually associated with excess amounts of serum uric acid which may lead to the deposition of monosodium urate crystals in the joints and kidneys. Hyperuricemia is also regarded as an important risk factor for inflammation, gouty arthritis, uric acid nephrolithiasis, hypertension, cardiovascular and renal diseases, and metabolic syndrome [1–3]. Compounds that display the ability to inhibit the biosynthesis of uric acid or those that inhibit the renal inflammation have been employed for the treatment of gout in recent years [4,5]. Until to now, a greatly increasing number of research have been put emphasis on the inflammatory events involved in hyperuricemia. To begin with, NLRP3 inflammasome, a complex protein compound consisting of NLRP3, apoptosis-associated speck-like protein adaptor (ASC) and pro-caspase-1, is recognized as an essential factor involved in the pathogenesis of many renal diseases and their complications. Activation of NLRP3 mediates pro-caspase-1 into its active form, which is important for the
* Corresponding author. E-mail address: [email protected] (W. Suling).
maturation of IL-1b and IL-18 in response to exogenous or endogenous stimuli. Evidences demonstrate that NLRP3-mediated inflammation caused by uric acid play a crucial role during renal injury in hyperuricemic rodents and patients [6,7]. Previous research also exhibits that inhibition of NLRP3 inflammasome alleviates the renal inflammation and ameliorates the renal ischemia-reperfusion injury [8]. Another finding shows that NLRP3 inflammasome activation participates in the pathogenesis of acute and chronic renal injury [9]. In addition, IL-1b maturation in response to various stimuli requires another signal pathway, NF-kB pathway. NLRP3 inflammasome activation and NF-kB pathway sensitization make the combined action on regulating IL-1b transcription and function. Therefore, we hypothesize that the interventions of NLRP3 inflammasome and NF-kB pathway might inhibit the production of inflammatory cytokines, and consequently alleviate potassium oxonate-induced renal injury. The aquaporins (AQPs) are water channel proteins that have been reported to be involved in the renal diseases. Renal function is mainly modulated by 7 AQP subtypes, namely AQP1, 2, 3, 4, 6, 7 and 8. The expression of AQP 2, 3 and 4 may be changed during dehydration or water overload [10]. Currently, it is believed that AQP 4 plays an important role in inflammatory responses. AQP 4 is reported to mediate pro-inflammatory factors in the stressinduced renal insufficiency through the regulation of endoplasmic
http://dx.doi.org/10.1016/j.biopha.2016.11.009 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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L. Hongyan et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
reticulum stress [11]. Up-regulated AQP 4 and water permeability by TLR4 and CRH/CRHR1 signaling also aggravates systemic inflammation and elicits cerebral edema [12]. Moreover, research shows that AQP 4 modulates communication between astrocyte and microglia communication in neuroinflammation [13]. Although a great number of findings demonstrate the impact of AQP 4 on inflammatory diseases, the role of AQP4 has not yet been introduced in potassium oxonate-induced hyperuricemic rats. Liquiritigenin, one of the flavonoid compounds extracted from Glycyrrhizae Radix et Rhizoma, possesses kinds of healthy properties including antioxidation, anti-inflammation, antidiabetes, cardioprotection, and neuroprotection. Some researchers report the effects of liquiritigenin on renal dysfunction recently. For instance, liquiritigenin and its glucuronides exhibit therapeutic effects on acute renal failure induced by uranyl nitrate [14]. Liquiritigenin plays protective action in the proliferation, apoptosis and invasion of renal cell carcinoma [15]. Others also show the anti-inflammatory activities of liquiritigenin. Licorice extract and liquiritigenin may inhibit the expression of pro-inflammatory mediators [16]. Liquiritigenin also suppresses inflammation via inhibition of NF-kB and MAPK signaling pathways in lipopolysaccharide-stimulated macrophage cells [17]. Based on the investigations above-mentioned, the present study is designed to explore the antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats and possible mechanisms for this action. 2. Materials and methods 2.1. Reagents
hyperuricemia rats treated with liquiritigenin (20 mg/kg); (5) the hyperuricemia rats treated with liquiritigenin (40 mg/kg). Potassium oxonate were dissolved in distilled water. Mice were treated once daily with potassium oxonate (250 mg/kg) or water (control) by gavage for 7 days. Liquiritigenin and allopurinol were administrated to the rats orally 1 h after the potassium oxonate exposure. 2.5. Determination of uric acid in serum and urine and creatinine clearance Samples of blood, urine and kidney tissues were collected 1 h after the last administration on the seventh day. The blood clotted for approximately 1 h at room temperature and centrifuged at 5,000g for 10 min to obtain serum sample. Uric acid concentrations in urine and serum were determined by the phosphotungstic acid method as previous described [18]. The creatinine clearance (Ccr) reflected to glomerularfiltration rate (GFR) was calculated using the following equation: Ccr(mi/min) = (urine creatinine urine flow rate)/serum creatinine 2.6. Determination of proinflammatory cytokines in serum and kidney Levels of IL-1b, IL-6, TNF-a in serum and kidney were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing KeyGEN Biotech. CO., LTD., Nanjing, China) according to the manufacturer's instructions.
Potassium oxonate and allopurinol were purchased from Sigma (St. Louis, MO, USA). Liquiritigenin was purchased from Shanghai pureone biotechnology Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of IL-1b, IL-6 and TNF-a were obtained by Nanjing KeyGEN Biotech. CO., Ltd. (Nanjing, China). Primary antibodies against AQP4, p-NF-kBp65, NF-kBp65, p-IkBa, IkBa, NLRP3, ASC, Caspase-1, IL-1b were produced by Cell Signaling Technology (Danvers, USA).
After the sacrifice of the rats, renal tissues were fixed in buffered neutral 10% formalin and dehydrated with increasing concentrations of ethanol, and then the tissues were embedded in paraffin and sectioned at 5 mm for staining with haematoxylin and eosin. The prepared sections were visualized under light microscopy (Nikon, Tokyo, Japan) at 200 magnifications.
2.2. Ethics statement
2.8. Western blot analysis
The animals received care in accordance with the guide for the care and the use of laboratory animals. Animal experimentation and the corresponding protocol (No.: 00162143; Date: 28/2/2016) were approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Changchun, China). All the procedures were in strict accordance with the P.R. China legislation on the use and care of laboratory animals.
Renal tissues were homogenized in ice-cold RIPA buffer (0.1% phenylmethylsulfonyl fluoride) and then centrifugated at 12000 g for 20 min. The protein concentration of supernatant was determined by BCA protein assay kit (Beyotime Biotechnology, Nanjing, China). The samples were loaded by a SDS-polyacrylamide gel electrophoresis and the resolved proteins were transferred onto PVDF membranes. After that, the PVDF membranes were blocked with 5% skim milk and incubated with separate primary antibodies at 4 C overnight. The primary antibodies include antiAQP4, anti-p-NF-kB p65, anti-NF-kB p65, anti-p-IkBa, anti-IkBa, anti-NLRP3, anti-ASC, anti-caspase-1, anti-IL-1b (CST, Danvers, USA) and anti-GAPDH (inner control). The membranes were finally incubated with secondary antibody at room temperature after washing with Tris-buffered saline–tween 20. The bands were visualized by a gel imaging system using chemiluminescence detection reagents (Beyotime Biotechnology, Nanjing, China) and densitometric analysis were performed by Image J software (National Institutes of Health, USA).
2.3. Animals Sprague Dawley rats (220 20 g, male and female in half) were purchased from the Laboratory Animal Center of Changchun University of Chinese Medicine, Changchun, China. Animals were housed under room temperature and had free access to food and water. Animal handling was in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation.
2.7. Histopathological examination
2.4. Induction of hyperuricemia and experimental design 2.9. Statistical analysis Rats were randomly divided into five groups: (1) control group; (2) model group (toxin control group): the rats were administered with potassium oxonate (250 mg/kg) to induce hyperuricemia; (3) the hyperuricemia rats treated with allopurinol (5 mg/kg); (4) the
Data were expressed as mean S.E.M. Results were analyzed by analysis of variance (ANOVA) with Tukey's post hoc test, by SPSS 17.0 (SPSS Inc., USA). P value < 0.05 was considered to be significant.
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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3. Results 3.1. Liquiritigenin decreased the level of uric acid in serum and urine of hyperuricemic rats Fig. 1 showed the effects of liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/kg) administration on levels of uric acid in serum and urine in hyperuricemic and normal rats. Potassium oxonate administration significantly elevated levels of uric acid in serum and urine compared with normal rats. Liquiritigenin at doses of 20, 40 mg/kg significantly decreased uric acid levels both in serum and urine. Allopurinol (5 mg/kg) as a positive control reduced the levels of uric acid in hyperuricemic rats. 3.2. Liquiritigenin inhibited the production of proinflammatory cytokines in serum and kidney in hyperuricemic rats As expected, levels of inflammatory cytokines IL-1b, IL-6 and TNF-a in serum and kidney showed a notable augment in potassium oxonate-induced hyperuricemic rats, compared with the control group. Liquiritigenin (20, 40 mg/kg) treatment significantly reversed these elevations in serum and renal tissue induced by potassium oxonate. Allopurinol (5 mg/kg) administration also restored the potassium oxonate-induced elevation of IL-1b, IL-6 and TNF-a both in serum and renal tissue (Fig. 2A–F). 3.3. Effects of liquiritigenin treatment on histological changes Renal histological changes between the normal control rats and experimental rats were depicted in Fig. 3. Renal sections from the control treated rats showed normal renal architecture, while
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potassium oxonate-treated rats displayed inflammatory cell infiltration in renal interstitium and disappearance of brush border epithelial cells with shrunken renal tubules. These results were diminished significantly by liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/kg). 3.4. Liquiritigenin improves renal inflammation by suppressing renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome activation in hyperuricemic rats As shown in Fig. 4, renal dysfunction was observed in hyperuricemic rats induced by potassium oxonate. Increased level of AQP 4 protein was observed in hyperuricemic rats, while Liquiritigenin (20, 40 mg/kg) down-regulated this level in the kidney of potassium oxonate-induced rats. Allopurinol (5 mg/kg) administration also inhibited the activation of AQP4 induced by potassium oxonate exposure. Moreover, the level of nuclear NF-kB p65 was increased by potassium oxonate treatment, and this effect was attenuated by liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) (Fig. 5). Thirdly, the phosphorylation of IkBa induced by potassium oxonate was reduced by the treatment with liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg). These results suggest that liquiritigenin may exert the inhibitory action by suppressing activated NF-kB/IkBa signaling pathway. Finally, elevated levels of inflammasome components (NLRP3, ASC, and caspase-1) and IL-1b in the renal tissues of potassium oxonateinduced rats were significantly attenuated by liquiritigenin (20, 40 mg/kg). Allopurinol (5 mg/kg) treatment was also associated with reduced protein expression of NLRP3, ASC, and caspase-1 and IL-1b in hyperuricemic rats when compared with model group.
Fig. 1. Effects of liquiritigenin on serum, urine uric acid level and creatinine clearance in potassium oxonate-induced hyperuricemic rats. Values are expressed as means S.E. M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.001; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 2. Effects of liquiritigenin on pro-inflammatory cytokines IL-1b (A), IL-6 (B) and TNF-a (C) in serum and IL-1b (D), IL-6 (E) and TNF-a (F) in renal tissues of potassium oxonate-induced hyperuricemic rats. Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.001; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3. Effects of liquiritigenin on renal histopathological examination (x = 200) in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonateinduced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). The black arrows represent inflammatory infiltration.
4. Discussion and conclusion Liquiritigenin has previously been shown the therapeutic effects on renal diseases. In the present study, we reported for
the first time that liquiritigenin may alleviate hyperuricemia and has renal protective effects in potassium oxonate-induced hyperuricemic rats. Liquiritigenin suppresses the activation of renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome, resulting
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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Fig. 4. Effects of liquiritigenin on AQP4 mediated NF-kB inflammatory pathways by western blot analysis in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonate-induced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.01; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.01.
in the reduction of renal inflammation and hyperuricemia in this animal model. These findings suggest that liquiritigenin is a potential drug for the treatment of hyperunicemia and renal injury. Serum urate level is most often consequent to renal urate excretion. The present study revealed that liquiritigenin reduces
uric acid level in serum and urine. Liquiritigenin also significant reverses the elevated production of pro-inflammatory cytokines in serum and renal tissues. In addition, renal histological examinations of renal tissues exhibited severe necrosis and inflammatory cell infiltration in potassium oxonate-treated rats, while these
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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Fig. 5. Effects of liquiritigenin on NLRP3 inflammasome in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonate-induced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.01; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.01.
characteristics significantly attenuate by liquiritigenin or allopurinol. These results illustrate the anti-inflammatory activity of liquiritigenin may contribute to the anti-hyperunicemic and renal protective effects. However, these findings are not enough to explain the underlying mechanism of liquiritigenin in hyperuricemic rats. As inflammation has been implicated in potassium oxonatetreated rats, we further determined the expression of several inflammation-related renal proteins to elucidate the possible mechanism. As reported, the relationship between expression of AQPs and pro-inflammatory cytokines is extensively investigated in pulmonary edema [19]. However, it is little known about the action of renal AQP4 in hyperuricemic animals. We therefore examine whether AQP4 is associated with the potassium oxonate-
induced renal injury. In the present study, the overexpression of AQP4 was observed in potassium oxonate-induced animal model. Liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) may downregulate AQP4 at protein level in the renal tissue of hyperuricemic rats. Exploration of the underlying mechanisms involving in its action reveals that liquiritigenin inhibits the activation of AQP4 induced by potassium oxonate. In addition to the role of AQP 4 plays in hyperuricemia, previous study has showed that activation of NF-kB is involved in the induction of expression of AQP4 by IL-1b, and blockade of NF-kB pathway significantly inhibits the induction of AQP4 mRNA and protein by cytokines [20]. In order to characterize the specific contributions of signal transduction mechanisms in potassium oxonate-induced AQP4 expression, the effects of NF-kB activity
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were examined in our study. Our results show that expressions of NF-kB p65 and IkBa are significantly activated in potassium oxonate-induced hyperuricemic rats. Liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) may effectively inhibit the activation of NF-kB p65 and the degradation of IkBa in hyperuricemic rats. These results are consistent with previous findings that expression of AQP4 in response to stimuli is regulated by the NF-kB pathway, which may be a previously unidentified regulatory system for AQP4 expression. Yet another increasingly attracted mechanism involves in regulating inflammation is the NLRP3 inflammasome pathway. NLRP3 inflammasome is a multi-protein complex, consisting NLRP3, ASC adaptor and pro-caspase-1, which controls caspase1 activation required for the maturation of cytokine IL-1b and IL-18 [21]. Recent reports show that NLRP3 inflammasome, as a mediator, is involved in the pathogenesis of a number of renal diseases and their complications. Diet-induced activation and production of IL-1b and IL-18 give rise to renal dysfunction in mice, which may be attenuated by NLRP3 inflammasome inhibition [22]. NLRP3 inflammasome inhibition and Nrf2 up-regulation attenuate sepsis-induced acute renal injury [23]. Hyperuricemia-induced NLRP3 activation of macrophages is reported as the contributors to the progression of diabetic nephropathy [24]. Our results show that potassium oxonate obviously increases the expressions of NLRP3, ASC and cleaved caspased-1 in hyperuricemic animals. These alterations may be restored by liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg). Until these points, our finding illustrates that liquiritigenin might inhibit uric acid concentration and ameliorate renal inflammation by suppressing renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome activation in potassium oxonateinduced hyperuricemic rats. At the same time, however, much remains to be investigated. These and other questions should reward research in the years to come. Acknowledgment
[5] [6] [7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19] [20]
None. [21]
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Original article
Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats Long Hongyana , Wu Sulingb,* , Zhu Weinaa , Zhang Yajiea , Ruan Jiea a b
Central Laboratory, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing 210001, China Department of Rheumatology, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing 210001, China
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 25 October 2016 Accepted 1 November 2016 Keywords: Liquiritigenin Hyperuricemia AQP4 NLRP3 Inflammation
A B S T R A C T
The aim is to investigate the anti-hyperuricemic and renal protective effects of liquiritigenin in potassium oxonate-induced hyperuricemic rats. Hyperuricemia in rats was induced were induced with potassium oxonate (250 mg/kg) intragastrically for 7 days, and liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/ kg) were daily administrated to the rats orally 1 h after the potassium oxonate exposure. Liquiritigenin significantly reversed the elevated productions of uric acid in serum and urine and pro-inflammation cytokines in serum and kidney, which shown that liquiritigenin has renal protective effects. Histological study shows that liquiritigenin inhibited severe necrosis and inflammatory cell infiltration in potassium oxonate-treated rats. Furthermore, liquiritigenin mediated the activities of aquaporins 4 (AQP4), and regulated the activation of NF-kB p65 and the degradation of IkBa. Finally, significant increases of nodlike receptor protein 3 (NLRP3) inflammasome, apoptosis-associated speck-like protein adaptor (ASC) adaptor and cleaved caspased-1 were restored by liquiritigenin. Therefore, liquiritigenin might improve renal inflammation by suppressing renal AQP4/NF-kB/IkBa and NLRP3 inflammasome activation in hyperuricemic rats. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Hyperuricemia, a high-prevalence metabolic disease, is usually associated with excess amounts of serum uric acid which may lead to the deposition of monosodium urate crystals in the joints and kidneys. Hyperuricemia is also regarded as an important risk factor for inflammation, gouty arthritis, uric acid nephrolithiasis, hypertension, cardiovascular and renal diseases, and metabolic syndrome [1–3]. Compounds that display the ability to inhibit the biosynthesis of uric acid or those that inhibit the renal inflammation have been employed for the treatment of gout in recent years [4,5]. Until to now, a greatly increasing number of research have been put emphasis on the inflammatory events involved in hyperuricemia. To begin with, NLRP3 inflammasome, a complex protein compound consisting of NLRP3, apoptosis-associated speck-like protein adaptor (ASC) and pro-caspase-1, is recognized as an essential factor involved in the pathogenesis of many renal diseases and their complications. Activation of NLRP3 mediates pro-caspase-1 into its active form, which is important for the
* Corresponding author. E-mail address: [email protected] (W. Suling).
maturation of IL-1b and IL-18 in response to exogenous or endogenous stimuli. Evidences demonstrate that NLRP3-mediated inflammation caused by uric acid play a crucial role during renal injury in hyperuricemic rodents and patients [6,7]. Previous research also exhibits that inhibition of NLRP3 inflammasome alleviates the renal inflammation and ameliorates the renal ischemia-reperfusion injury [8]. Another finding shows that NLRP3 inflammasome activation participates in the pathogenesis of acute and chronic renal injury [9]. In addition, IL-1b maturation in response to various stimuli requires another signal pathway, NF-kB pathway. NLRP3 inflammasome activation and NF-kB pathway sensitization make the combined action on regulating IL-1b transcription and function. Therefore, we hypothesize that the interventions of NLRP3 inflammasome and NF-kB pathway might inhibit the production of inflammatory cytokines, and consequently alleviate potassium oxonate-induced renal injury. The aquaporins (AQPs) are water channel proteins that have been reported to be involved in the renal diseases. Renal function is mainly modulated by 7 AQP subtypes, namely AQP1, 2, 3, 4, 6, 7 and 8. The expression of AQP 2, 3 and 4 may be changed during dehydration or water overload [10]. Currently, it is believed that AQP 4 plays an important role in inflammatory responses. AQP 4 is reported to mediate pro-inflammatory factors in the stressinduced renal insufficiency through the regulation of endoplasmic
http://dx.doi.org/10.1016/j.biopha.2016.11.009 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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reticulum stress [11]. Up-regulated AQP 4 and water permeability by TLR4 and CRH/CRHR1 signaling also aggravates systemic inflammation and elicits cerebral edema [12]. Moreover, research shows that AQP 4 modulates communication between astrocyte and microglia communication in neuroinflammation [13]. Although a great number of findings demonstrate the impact of AQP 4 on inflammatory diseases, the role of AQP4 has not yet been introduced in potassium oxonate-induced hyperuricemic rats. Liquiritigenin, one of the flavonoid compounds extracted from Glycyrrhizae Radix et Rhizoma, possesses kinds of healthy properties including antioxidation, anti-inflammation, antidiabetes, cardioprotection, and neuroprotection. Some researchers report the effects of liquiritigenin on renal dysfunction recently. For instance, liquiritigenin and its glucuronides exhibit therapeutic effects on acute renal failure induced by uranyl nitrate [14]. Liquiritigenin plays protective action in the proliferation, apoptosis and invasion of renal cell carcinoma [15]. Others also show the anti-inflammatory activities of liquiritigenin. Licorice extract and liquiritigenin may inhibit the expression of pro-inflammatory mediators [16]. Liquiritigenin also suppresses inflammation via inhibition of NF-kB and MAPK signaling pathways in lipopolysaccharide-stimulated macrophage cells [17]. Based on the investigations above-mentioned, the present study is designed to explore the antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats and possible mechanisms for this action. 2. Materials and methods 2.1. Reagents
hyperuricemia rats treated with liquiritigenin (20 mg/kg); (5) the hyperuricemia rats treated with liquiritigenin (40 mg/kg). Potassium oxonate were dissolved in distilled water. Mice were treated once daily with potassium oxonate (250 mg/kg) or water (control) by gavage for 7 days. Liquiritigenin and allopurinol were administrated to the rats orally 1 h after the potassium oxonate exposure. 2.5. Determination of uric acid in serum and urine and creatinine clearance Samples of blood, urine and kidney tissues were collected 1 h after the last administration on the seventh day. The blood clotted for approximately 1 h at room temperature and centrifuged at 5,000g for 10 min to obtain serum sample. Uric acid concentrations in urine and serum were determined by the phosphotungstic acid method as previous described [18]. The creatinine clearance (Ccr) reflected to glomerularfiltration rate (GFR) was calculated using the following equation: Ccr(mi/min) = (urine creatinine urine flow rate)/serum creatinine 2.6. Determination of proinflammatory cytokines in serum and kidney Levels of IL-1b, IL-6, TNF-a in serum and kidney were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing KeyGEN Biotech. CO., LTD., Nanjing, China) according to the manufacturer's instructions.
Potassium oxonate and allopurinol were purchased from Sigma (St. Louis, MO, USA). Liquiritigenin was purchased from Shanghai pureone biotechnology Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of IL-1b, IL-6 and TNF-a were obtained by Nanjing KeyGEN Biotech. CO., Ltd. (Nanjing, China). Primary antibodies against AQP4, p-NF-kBp65, NF-kBp65, p-IkBa, IkBa, NLRP3, ASC, Caspase-1, IL-1b were produced by Cell Signaling Technology (Danvers, USA).
After the sacrifice of the rats, renal tissues were fixed in buffered neutral 10% formalin and dehydrated with increasing concentrations of ethanol, and then the tissues were embedded in paraffin and sectioned at 5 mm for staining with haematoxylin and eosin. The prepared sections were visualized under light microscopy (Nikon, Tokyo, Japan) at 200 magnifications.
2.2. Ethics statement
2.8. Western blot analysis
The animals received care in accordance with the guide for the care and the use of laboratory animals. Animal experimentation and the corresponding protocol (No.: 00162143; Date: 28/2/2016) were approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Changchun, China). All the procedures were in strict accordance with the P.R. China legislation on the use and care of laboratory animals.
Renal tissues were homogenized in ice-cold RIPA buffer (0.1% phenylmethylsulfonyl fluoride) and then centrifugated at 12000 g for 20 min. The protein concentration of supernatant was determined by BCA protein assay kit (Beyotime Biotechnology, Nanjing, China). The samples were loaded by a SDS-polyacrylamide gel electrophoresis and the resolved proteins were transferred onto PVDF membranes. After that, the PVDF membranes were blocked with 5% skim milk and incubated with separate primary antibodies at 4 C overnight. The primary antibodies include antiAQP4, anti-p-NF-kB p65, anti-NF-kB p65, anti-p-IkBa, anti-IkBa, anti-NLRP3, anti-ASC, anti-caspase-1, anti-IL-1b (CST, Danvers, USA) and anti-GAPDH (inner control). The membranes were finally incubated with secondary antibody at room temperature after washing with Tris-buffered saline–tween 20. The bands were visualized by a gel imaging system using chemiluminescence detection reagents (Beyotime Biotechnology, Nanjing, China) and densitometric analysis were performed by Image J software (National Institutes of Health, USA).
2.3. Animals Sprague Dawley rats (220 20 g, male and female in half) were purchased from the Laboratory Animal Center of Changchun University of Chinese Medicine, Changchun, China. Animals were housed under room temperature and had free access to food and water. Animal handling was in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation.
2.7. Histopathological examination
2.4. Induction of hyperuricemia and experimental design 2.9. Statistical analysis Rats were randomly divided into five groups: (1) control group; (2) model group (toxin control group): the rats were administered with potassium oxonate (250 mg/kg) to induce hyperuricemia; (3) the hyperuricemia rats treated with allopurinol (5 mg/kg); (4) the
Data were expressed as mean S.E.M. Results were analyzed by analysis of variance (ANOVA) with Tukey's post hoc test, by SPSS 17.0 (SPSS Inc., USA). P value < 0.05 was considered to be significant.
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3. Results 3.1. Liquiritigenin decreased the level of uric acid in serum and urine of hyperuricemic rats Fig. 1 showed the effects of liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/kg) administration on levels of uric acid in serum and urine in hyperuricemic and normal rats. Potassium oxonate administration significantly elevated levels of uric acid in serum and urine compared with normal rats. Liquiritigenin at doses of 20, 40 mg/kg significantly decreased uric acid levels both in serum and urine. Allopurinol (5 mg/kg) as a positive control reduced the levels of uric acid in hyperuricemic rats. 3.2. Liquiritigenin inhibited the production of proinflammatory cytokines in serum and kidney in hyperuricemic rats As expected, levels of inflammatory cytokines IL-1b, IL-6 and TNF-a in serum and kidney showed a notable augment in potassium oxonate-induced hyperuricemic rats, compared with the control group. Liquiritigenin (20, 40 mg/kg) treatment significantly reversed these elevations in serum and renal tissue induced by potassium oxonate. Allopurinol (5 mg/kg) administration also restored the potassium oxonate-induced elevation of IL-1b, IL-6 and TNF-a both in serum and renal tissue (Fig. 2A–F). 3.3. Effects of liquiritigenin treatment on histological changes Renal histological changes between the normal control rats and experimental rats were depicted in Fig. 3. Renal sections from the control treated rats showed normal renal architecture, while
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potassium oxonate-treated rats displayed inflammatory cell infiltration in renal interstitium and disappearance of brush border epithelial cells with shrunken renal tubules. These results were diminished significantly by liquiritigenin (20, 40 mg/kg) and allopurinol (5 mg/kg). 3.4. Liquiritigenin improves renal inflammation by suppressing renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome activation in hyperuricemic rats As shown in Fig. 4, renal dysfunction was observed in hyperuricemic rats induced by potassium oxonate. Increased level of AQP 4 protein was observed in hyperuricemic rats, while Liquiritigenin (20, 40 mg/kg) down-regulated this level in the kidney of potassium oxonate-induced rats. Allopurinol (5 mg/kg) administration also inhibited the activation of AQP4 induced by potassium oxonate exposure. Moreover, the level of nuclear NF-kB p65 was increased by potassium oxonate treatment, and this effect was attenuated by liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) (Fig. 5). Thirdly, the phosphorylation of IkBa induced by potassium oxonate was reduced by the treatment with liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg). These results suggest that liquiritigenin may exert the inhibitory action by suppressing activated NF-kB/IkBa signaling pathway. Finally, elevated levels of inflammasome components (NLRP3, ASC, and caspase-1) and IL-1b in the renal tissues of potassium oxonateinduced rats were significantly attenuated by liquiritigenin (20, 40 mg/kg). Allopurinol (5 mg/kg) treatment was also associated with reduced protein expression of NLRP3, ASC, and caspase-1 and IL-1b in hyperuricemic rats when compared with model group.
Fig. 1. Effects of liquiritigenin on serum, urine uric acid level and creatinine clearance in potassium oxonate-induced hyperuricemic rats. Values are expressed as means S.E. M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.001; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 2. Effects of liquiritigenin on pro-inflammatory cytokines IL-1b (A), IL-6 (B) and TNF-a (C) in serum and IL-1b (D), IL-6 (E) and TNF-a (F) in renal tissues of potassium oxonate-induced hyperuricemic rats. Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.001; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3. Effects of liquiritigenin on renal histopathological examination (x = 200) in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonateinduced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). The black arrows represent inflammatory infiltration.
4. Discussion and conclusion Liquiritigenin has previously been shown the therapeutic effects on renal diseases. In the present study, we reported for
the first time that liquiritigenin may alleviate hyperuricemia and has renal protective effects in potassium oxonate-induced hyperuricemic rats. Liquiritigenin suppresses the activation of renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome, resulting
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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Fig. 4. Effects of liquiritigenin on AQP4 mediated NF-kB inflammatory pathways by western blot analysis in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonate-induced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.01; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.01.
in the reduction of renal inflammation and hyperuricemia in this animal model. These findings suggest that liquiritigenin is a potential drug for the treatment of hyperunicemia and renal injury. Serum urate level is most often consequent to renal urate excretion. The present study revealed that liquiritigenin reduces
uric acid level in serum and urine. Liquiritigenin also significant reverses the elevated production of pro-inflammatory cytokines in serum and renal tissues. In addition, renal histological examinations of renal tissues exhibited severe necrosis and inflammatory cell infiltration in potassium oxonate-treated rats, while these
Please cite this article in press as: L. Hongyan, et al., Antihyperuricemic effect of liquiritigenin in potassium oxonate-induced hyperuricemic rats, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.009
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Fig. 5. Effects of liquiritigenin on NLRP3 inflammasome in potassium oxonate-induced hyperuricemic rats. (A) Control; (B) Potassium oxonate-induced hyperuricemia; (C) Potassium oxonate + liquiritigenin (20 mg/kg); (D) Potassium oxonate + liquiritigenin (40 mg/kg); (E) Potassium oxonate + allopurinol (5 mg/kg). Values are expressed as means S.E.M. Compared with control group: #P < 0.05, ##P < 0.01, ###P < 0.01; Compared with model group: *P < 0.05, **P < 0.01, ***P < 0.01.
characteristics significantly attenuate by liquiritigenin or allopurinol. These results illustrate the anti-inflammatory activity of liquiritigenin may contribute to the anti-hyperunicemic and renal protective effects. However, these findings are not enough to explain the underlying mechanism of liquiritigenin in hyperuricemic rats. As inflammation has been implicated in potassium oxonatetreated rats, we further determined the expression of several inflammation-related renal proteins to elucidate the possible mechanism. As reported, the relationship between expression of AQPs and pro-inflammatory cytokines is extensively investigated in pulmonary edema [19]. However, it is little known about the action of renal AQP4 in hyperuricemic animals. We therefore examine whether AQP4 is associated with the potassium oxonate-
induced renal injury. In the present study, the overexpression of AQP4 was observed in potassium oxonate-induced animal model. Liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) may downregulate AQP4 at protein level in the renal tissue of hyperuricemic rats. Exploration of the underlying mechanisms involving in its action reveals that liquiritigenin inhibits the activation of AQP4 induced by potassium oxonate. In addition to the role of AQP 4 plays in hyperuricemia, previous study has showed that activation of NF-kB is involved in the induction of expression of AQP4 by IL-1b, and blockade of NF-kB pathway significantly inhibits the induction of AQP4 mRNA and protein by cytokines [20]. In order to characterize the specific contributions of signal transduction mechanisms in potassium oxonate-induced AQP4 expression, the effects of NF-kB activity
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were examined in our study. Our results show that expressions of NF-kB p65 and IkBa are significantly activated in potassium oxonate-induced hyperuricemic rats. Liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg) may effectively inhibit the activation of NF-kB p65 and the degradation of IkBa in hyperuricemic rats. These results are consistent with previous findings that expression of AQP4 in response to stimuli is regulated by the NF-kB pathway, which may be a previously unidentified regulatory system for AQP4 expression. Yet another increasingly attracted mechanism involves in regulating inflammation is the NLRP3 inflammasome pathway. NLRP3 inflammasome is a multi-protein complex, consisting NLRP3, ASC adaptor and pro-caspase-1, which controls caspase1 activation required for the maturation of cytokine IL-1b and IL-18 [21]. Recent reports show that NLRP3 inflammasome, as a mediator, is involved in the pathogenesis of a number of renal diseases and their complications. Diet-induced activation and production of IL-1b and IL-18 give rise to renal dysfunction in mice, which may be attenuated by NLRP3 inflammasome inhibition [22]. NLRP3 inflammasome inhibition and Nrf2 up-regulation attenuate sepsis-induced acute renal injury [23]. Hyperuricemia-induced NLRP3 activation of macrophages is reported as the contributors to the progression of diabetic nephropathy [24]. Our results show that potassium oxonate obviously increases the expressions of NLRP3, ASC and cleaved caspased-1 in hyperuricemic animals. These alterations may be restored by liquiritigenin (20, 40 mg/kg) or allopurinol (5 mg/kg). Until these points, our finding illustrates that liquiritigenin might inhibit uric acid concentration and ameliorate renal inflammation by suppressing renal AQP4/NF-kB/IkBa signaling and NLRP3 inflammasome activation in potassium oxonateinduced hyperuricemic rats. At the same time, however, much remains to be investigated. These and other questions should reward research in the years to come. Acknowledgment
[5] [6] [7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19] [20]
None. [21]
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