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Akt pathway in human proximal tubular epithelial cells
Biomedicine & Pharmacotherapy 106 (2018) 1175–1181
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Astilbin inhibits high glucose-induced autophagy and apoptosis through the PI3K/Akt pathway in human proximal tubular epithelial cells Fang Chen
⁎,1
T
, Zhiqiang Sun1, Xiaoguang Zhu, Yali Ma
Department of Nephrology, Huaihe Hospital of Henan University, Kaifeng 475000, Henan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Diabetic nephropathy (DN) Astilbin Autophagy Cell apoptosis PI3K/Akt pathway
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. It has been found that astilbin, a flavonoid compound, exerts a protective effect on DN. However, the role of astilbin in autophagy during DN is unknown. The human proximal tubular epithelial cells (HK-2 cells) were treated with high glucose (HG, 30 mM) in the presence or absence of astilbin. Cell viability was measured by MTT assay. The autophagy was determined by detecting the expression of LC3-II and p62 using western blot. The cell apoptosis was evaluated by detecting the apoptosis rate, caspase-3 activity, and the expression of Bcl-2 and Bax. The expression levels of protein kinase B (Akt) and p-Akt were detected by western blot. To determine whether the phosphatidylinositol-3-kinase (PI3K)/Akt pathway was involved in the effect of astilbin, cells were treated with the inhibitor of Akt, LY294002. We found that astilbin (10 and 20 μM) did not affect the viability of HK-2 cells, but attenuated HG-induced cell viability. Astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells. The expression of p-Akt was inhibited by HG treatment, while the inhibitory effect of HG was attenuated by astilbin. Inhibition of the PI3K/ Akt signaling resisted the effect of astilbin on HG-induced apoptosis and autophagy. In conclusion, astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells through the PI3K/Akt pathway. The results indicated that astilbin might be a new therapeutic agent and be useful for improving clinical management of DN.
1. Introduction Diabetic nephropathy (DN) is a devastating complication of diabetes mellitus and accounts for a significant increase in morbidity and mortality in patients with diabetes [1]. It is well known that DN is the leading cause of end-stage renal disease and is becoming a serious health problem worldwide [1]. Currently, the therapies for DN focus on controlling blood glucose levels and blood pressure, and regulating the renin-angiotensin system (RAS) to inhibit the development and progression of DN [2,3]. Therefore, it is critically needed to further understand the pathogenesis of DN to identify new therapies for DN. Autophagy is a part of the catabolic process that degrades the damaged proteins and organelles, maintaining cell homeostasis under various stress conditions [4,5]. Accumulating evidence suggests that dysregulated autophagy plays pathogenic roles in various diseases [6]. The functional roles of autophagy in the kidney under normal and disease conditions have been gradually clarified. Autophagy protects kidney from induction of aging [7], hypoxia [8], and anticancer drugs [9,10]. Therefore, regulating autophagy activity in DN may be usually
for the management of DN. Astilbin (Fig. 1A) is a flavonoid compound and possesses a variety of activities, such as anti-inflammatory and immunoregulatory effects [11,12]. Diao et al. [13] reported that astilbin protected diabetic rat heart against ischemia-reperfusion (I/R) injury via inhibiting the HMGB1-dependent NF-κB signaling pathway. Astilbin also exhibited anti-hyperglycaemic effect by inhibiting the intact microsomal glucose6-phosphatase [14]. Moreover, astilbin also exerted a protective effect on DN [15], suggesting astilbin might be a potential agent in DN therapy. However, the role of astilbin in autophagy during DN is unknown. In this study, we investigated the effect of astilbin on autophagy and apoptosis in high glucose (HG)-induced human proximal tubular epithelial cells (HK-2 cells). We found that astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells.
Abbreviations: AKT, protein kinase B; DN, diabetic nephropathy; HG, high glucose; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol-3-kinase ⁎ Corresponding author at: Department of Nephrology, Huaihe Hospital of Henan University, No. 115 Ximen Street, Kaifeng 475000, Henan, PR China. E-mail address: [email protected] (F. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2018.07.072 Received 13 April 2018; Received in revised form 7 July 2018; Accepted 13 July 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Effect of astilbin on cell viability in HG-induced cells. (A) Chemical structure of astilbin. (B) Cell viability of HK-2 cells treated with different concentrations of glucose (5.5, 10, 20, and 30 mM). *P < 0.05 vs. cells without glucose treatment. (C and D) Effect of astilbin (10 and 20 μg/ml) on the viability of HK-2 cells treated with low concentration of glucose (5.5 mM) and high concentration of glucose (HG, 30 mM). *P < 0.05 vs. cells without treatment of glucose or astilbin. # P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
Fig. 2. Effect of astilbin on HG-induced autophagy in HK-2 cells. HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. The expression levels of LC3-II and p62 were detected by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
2. Materials and methods
2.2. MTT assay
2.1. Cell culture
HK-2 cells (1 × 104cells/well) were seeded in 96-well plates, and cultured for 24 h. Then 0.5 mg/ml of MTT solution was added to each well and incubated for another 4 h at 37 °C. Subsequently, the formazan crystals were dissolved by adding DMSO solution. Finally, the absorbance was measured at the wavelength of 570 nm using a Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).
HK-2 cells were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China). The cells were maintained in the DF12 medium (Gibco Laboratories, NY, USA) containing 10% FBS and cultured in a humidified incubator at 37 °C with 5% CO2. HK-2 cells were treated with appointed concentrations of HG, astilbin (Shanghai Tauto Biotech Co., Ltd., Shanghai, China; purity > 98%), or/and LY294002 (the inhibitor of Akt; Sigma-Aldrich, St. Louis, MO, USA) for 24 h.
2.3. Western blot HK-2 cell extracts were obtained using lysis buffer (Invitrogen, Carlsbad, CA, USA). Proteins in the cell extracts were separated by 12% SDS-PAGE gels. The concentration of the protein was measured by 1176
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Fig. 3. Effect of astilbin on HG-induced apoptosis of HK-2 cells. HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. (A) The apoptosis rate was detected by flow cytometry. (B) Detection of caspase-3 activity. (C) The expression levels of Bcl-2 and Bax were determined by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
2.4. Flow cytometry
using a BCA protein assay kit (Pierce Biotechnology, IL, USA). The proteins were transferred to nitrocellulose membranes and the membranes were blocked with 5% non-fat milk in TBST buffer at 37 °C for 1 h. The membranes were then incubated with primary antibodies against LC3, p62, Bcl-2, Bax, Akt, p-Akt, and β-actin (dilution 1: 500, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then the proteins were probed by incubating with HRP-conjugated secondary antibody (dilution 1: 2000, Abcam) for 2 h at 37 °C. Finally, the brands were detected with ECL reagent (Millipore, Billerica, MA, USA).
The treated HK-2 cells were harvested and stained using a Annexin V-PI Apoptosis Detection Kit (Abcam). After incubation for 30 min in the dark, the cell apoptosis ratio was measured using FACScan flow cytometer (Becton Dickenson, San Jose, CA, USA). 2.5. Detection of caspase-3 activity HK-2 cell extracts containing 50 μg of proteins were incubated with 1177
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Fig. 4. Effect of astilbin on the PI3K/Akt pathway under HG condition. To evaluate whether the PI3K/Akt pathway was affected by astilbin under HG condition, HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. The expression levels of p-Akt and Akt were measured by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
Fig. 5. Effect of LY294002 on astilbin-induced autophagy inhibition under HG condition. To evaluate whether the PI3K/Akt pathway was involved in the effect of astilbin on cell autophagy under HG condition, HK-2 cells were treated with HG (30 mM) in the presence or absence of astilbin (20 μg/ml) or the inhibitor of Akt (LY294002, 10 μM) for 24 h. The expression levels of LC3-II and p62 were detected by western blot. *P < 0.05 vs. cells treated with HG alone. #P < 0.05 vs. cells treated with HG and astilbin. Significance was determined by one-way ANOVA. Glu: glucose. LY: LY294002.
100 μM of the enzyme-specific colorimetric substrate Ac-DEVD-pNA at 37 °C for 2 h. The colorimetric release of p-nitroaniline was determined by detecting the absorbance at the wavelength of 405 nm.
3.2. Astilbin inhibited HG-induced autophagy in HK-2 cells The amount of LC3-II, the LC3-II/LC3-I ratio or LC3-II/(LC3I + LC3-II) ratio is now widely used to monitor autophagy. Because LC3-II exhibits more sensitive than LC3-I detected using western blot, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3II, may not be appropriate, and rather, comparison of the LC3-II amount among different groups is likely to be a more accurate method to reflect the autophagy [16]. In the current study, the expression level of LC3-II was detected by western blot. As shown in Fig. 2, we found that HG induced LC3-II expression, and astilbin treatment (10 and 20 μg/ml) attenuated the induction. The nucleoporin p62 (p62) is a protein complex that is down-regulated during autophagy and is widely used to monitor autophagic activity [17,18]. In Fig. 2, HG inhibited p62 expression, while astilbin treatment (10 and 20 μg/ml) attenuated the inhibition. The results indicated that astilbin attenuated HG-induced autophagy in HK-2 cells.
2.6. Statistical analysis Data are shown as mean ± SD of three independent experiments. Comparison was assessed by one-way analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Chicago, IL, USA). A difference was considered significant when p value was less than 0.05.
3. Results 3.1. Astilbin inhibited the viability of HK-2 cells under HG condition To determine the effect of glucose on cell viability, HK-2 cells were cultured with different concentrations of glucose (5.5, 10, 20, and 30 mM). The results in Fig. 1B showed that high concentration of glucose (30 mM) induced the viability of HK-2 cells, while the low concentrations of glucose (5.5, 10, and 20 mM) did not affect the cell viability. To evaluate the effect of astilbin on HG-induced viability, cells were cultured with 5.5 or 30 mM of glucose in the presence or absence of 10 or 20 μg/ml astilbin for 24 h. The results in Fig. 1C and D indicated that astilbin attenuated the effect of HG (30 mM) on cell viability, however, astilbin did not affect the viability of cells treated with low concentration of glucose (5.5 mM).
3.3. Astilbin inhibited HG-induced apoptosis in HK-2 cells To further investigate the role of astilbin in HG-induced cell apoptosis, the apoptosis rate was detected by flow cytometry. The apoptosis rate was increased in cells treated with HG, and astilbin decreased the apoptosis rate under HG condition (Fig. 3A). The caspase-3 activity was also measured. The results in Fig. 3B showed that HG increased caspase3 activity, but astilbin treatment decreased the caspase-3 activity compared to cells treated with HG. The expression levels of Bcl-2 and Bax were determined by western blot. The Bcl-2 expression was 1178
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Fig. 6. Effect of LY294002 on astilbin-induced apoptosis inhibition under HG condition. To determine whether the PI3K/Akt pathway was involved in the effect of astilbin on cell apoptosis under HG condition, HK-2 cells were treated with HG (30 mM) in the presence or absence of astilbin (20 μg/ml) or the inhibitor of Akt (LY294002, 10 μM) for 24 h. (A) The apoptosis rate was detected by flow cytometry. (B) Detection of caspase-3 activity. (C) The expression levels of Bcl-2 and Bax were determined by western blot. *P < 0.05 vs. cells treated with HG alone. #P < 0.05 vs. cells treated with HG and astilbin. Significance was determined by oneway ANOVA. Glu: glucose. LY: LY294002.
3.6. The PI3K/Akt signaling was involved in the role of astilbin on HGinduced apoptosis
decreased and Bax expression was increased in the cells treated with HG, however, when the cells were treated with astilbin, the Bcl-2 expression was increased and Bax expression was decreased compared with cells only treated with HG (Fig. 3C). The results suggested that astilbin inhibited HG-induced apoptosis of HK-2 cells.
To determine whether the PI3K/Akt pathway was involved in the effect of astilbin on cell apoptosis, cells were incubated with LY294002 and the cell apoptosis was measured. As shown in Fig. 6A and B, the apoptosis rate and caspase-3 activity were decreased after treatment with astilbin under HG condition, but LY294002 reversed the effects of astilbin on the apoptosis rate and caspase-3 activity. The expression level of Bcl-2 was increased and the expression level of Bax was decreased in cells treated with astilbin under HG condition, but LY294002 reversed the effects of astilbin on Bcl-2 and Bax expression (Fig. 6C). The results indicated that the PI3K/Akt signaling was involved in the effect of astilbin on HG-induced cell apoptosis.
3.4. Astilbin activated the PI3K/Akt pathway under HG condition To evaluate whether the PI3K/Akt pathway was affected by astilbin under HG condition, the expression levels of p-Akt and Akt were measured by western blot. As shown in Fig. 4, expression of p-Akt was inhibited by HG treatment, while the inhibitory effect of HG was attenuated by astilbin. The treatment with HG and/or astilbin showed no significant effect on the expression of Akt, indicating that astilbin activated the PI3K/Akt pathway under HG condition.
4. Discussion 3.5. The PI3K/Akt signaling was involved in the role of astilbin on HGinduced autophagy
Elevated glucose level is one of the main risk factors for the development and progression of DN [19]. High blood sugar may lead to the formation of advanced glycation end products, which induce inflammation in the kidney and promote the development of DN [19]. HG is usually used for the establishment of in vitro model of DN [20,21]. Previous studies indicated that treatment with HG showed significant reduction in cell viability of HK-2 cells and induced renal tubular epithelial injury [22,23]. In our work, we found that HG (30 mM) inhibited the viability of HK-2 cells, while the low concentrations of glucose (5.5, 10, and 20 mM) did not affect the cell viability.
To evaluate whether the PI3K/Akt pathway was involved in the effect of astilbin on autophagy, cells were incubated with the inhibitor of Akt (LY294002) and the autophagy level was measured. As shown in Fig. 5, astilbin reduced the LC3-II/LC3-I ratio and increased p62 expression level under HG condition. However, LY294002 reversed astilbin-induced inhibitory effect on autophagy under HG condition. The results suggested that the PI3K/Akt signaling was involved in the effect of astilbin on HG-induced autophagy. 1179
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References
Autophagy plays a critical role in degrading cellular protein aggregates and damaged organelles, promoting cell survival and maintaining homeostasis. However, excessive autophagy can also contribute to apoptosis. Thus, autophagy may be deleterious. [24]. It was reported that within 24 h of exposure to HG condition induced autophagy in podocytes, suggesting that the acute exposure to HG could induce autophagy [25]. Administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys, and in cultured renal proximal tubular cells, cisplatin induced autophagy and apoptosis [26]. In the present study, we found that HG induced LC3-II expression, inhibited p62 expression, and increased the apoptosis rate in HK-2 cells, suggesting that HG induced autophagy and apoptosis in HK-2 cells. However, astilbin attenuated the induction effect of HG on autophagy and apoptosis in HK-2 cells. Zhao et al. [27] reported that liraglutide suppressed HG-induced autophagy and apoptosis through glucagon-like peptide-1 receptor in renal tubular epithelial cells. However, some studies displayed different results. A recent study showed that HG induced apoptosis via up-regulation of Bim expression, but not brought a significant change in autophagy in HK-2 cells. Knockdown of Bim inhibited HG-induced apoptosis and also sensitized HK-2 cells to autophagy under HG condition [28]. Curcumin reversed advanced glycation or glycoxidation end-product (AGE)-induced apoptosis by promoting autophagy through the PI3K/Akt signaling pathway in NRK-52E rat renal tubular epithelial cells [29]. These inconsistent findings may result from the different cell lines used. The PI3K/Akt pathway is a main intracellular signaling pathway that regulates multiple biological processes, such as cell proliferation, differentiation, and apoptosis under the physiological and pathological conditions [30]. It has been demonstrated that the PI3K/Akt pathway is closely related to autophagy [31]. Recently, Li et al. [32] reported that honokiol, the main active constituent of Magnolia officinalis, induced autophagy and apoptosis of osteosarcoma cells through the PI3K/Akt/ mammalian target of rapamycin (mTOR) signaling pathway. Curcumin exhibited the antitumor effect via regulating cell apoptosis and autophagy in human lung cancer A549 cells through inhibiting the PI3K/Akt/ mTOR pathway [33]. These studies revealed the important role of the PI3K/Akt pathway in regulating cell apoptosis and autophagy. The PI3K/Akt pathway was also reported to play an important role in DN. Huang et al. reported that Notoginsenoside R1 treatment ameliorates podocyte injury in rats with DN through suppressing inflammation and apoptosis via the PI3K/Akt signaling pathway [34]. Silencing of TRB3 ameliorated diabetic tubule interstitial nephropathy via the PI3K/AKT signaling in diabetic rats, suggesting that silencing of TRB3 had a significant ameliorative effect on diabetic nephropathy in rats [35]. KCa3.1 knockdown alleviated dysfunctional tubular autophagy in diabetic nephropathy through the PI3K/Akt/mTOR signaling pathway [36]. In the present study, we also found that the PI3K/Akt signaling was involved in the role of astilbin on HG-induced autophagy and cell apoptosis. However, the findings were based on the in vitro study, an in vivo study is still needed.
[1] F.B. Hu, Globalization of diabetes: the role of diet, lifestyle, and genes, Diabetes Care 34 (2011) 1249–1257. [2] P. Ruggenenti, P. Cravedi, G. Remuzzi, The RAAS in the pathogenesis and treatment of diabetic nephropathy, Nat. Rev. Nephrol. 6 (2010) 319–330. [3] N.A. Calcutt, M.E. Cooper, T.S. Kern, A.M. Schmidt, Therapies for hyperglycaemiainduced diabetic complications: from animal models to clinical trials, Nat. Rev. Drug Discov. 8 (2009) 417–429. [4] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through cellular self-digestion, Nature 451 (2008) 1069–1075. [5] J.H. Hurley, B.A. Schulman, Atomistic autophagy: the structures of cellular selfdigestion, Cell 157 (2014) 300–311. [6] W. Xie, J. Zhou, Aberrant regulation of autophagy in mammalian diseases, Biol. Lett. 14 (2018). [7] B. Hartleben, M. Godel, C. Meyer-Schwesinger, S. Liu, T. Ulrich, S. Kobler, T. Wiech, F. Grahammer, S.J. Arnold, M.T. Lindenmeyer, C.D. Cohen, H. Pavenstadt, D. Kerjaschki, N. Mizushima, A.S. Shaw, G. Walz, T.B. Huber, Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice, J. Clin. Invest. 120 (2010) 1084–1096. [8] T. Kimura, Y. Takabatake, A. Takahashi, J.Y. Kaimori, I. Matsui, T. Namba, H. Kitamura, F. Niimura, T. Matsusaka, T. Soga, H. Rakugi, Y. Isaka, Autophagy protects the proximal tubule from degeneration and acute ischemic injury, J. Am. Soc. Nephrol. 22 (2011) 902–913. [9] C. Yang, V. Kaushal, S.V. Shah, G.P. Kaushal, Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells, Am. J. Physiol. Ren. Physiol. 294 (2008) F777–787. [10] G.P. Kaushal, V. Kaushal, C. Herzog, C. Yang, Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity, Autophagy 4 (2008) 710–712. [11] J. Yu, Z. Xiao, R. Zhao, C. Lu, Y. Zhang, Astilbin emulsion improves guinea pig lesions in a psoriasis-like model by suppressing IL-6 and IL-22 via p38 MAPK, Mol. Med. Rep. 17 (2017) 3789–3796. [12] Q.F. Meng, Z. Zhang, Y.J. Wang, W. Chen, F.F. Li, L.T. Yue, C.J. Zhang, H. Li, M. Zhang, C.C. Wang, P. Zhang, H. Chen, R.S. Duan, S.M. Sun, Y.B. Li, Astilbin ameliorates experimental autoimmune myasthenia gravis by decreased Th17 cytokines and up-regulated T regulatory cells, J. Neuroimmunol. 298 (2016) 138–145. [13] H. Diao, Z. Kang, F. Han, W. Jiang, Astilbin protects diabetic rat heart against ischemia-reperfusion injury via blockade of HMGB1-dependent NF-kappaB signaling pathway, Food Chem. Toxicol. 63 (2014) 104–110. [14] O. Estrada, M. Hasegawa, F. Gonzalez-Mujica, N. Motta, E. Perdomo, A. Solorzano, J. Mendez, B. Mendez, E.G. Zea, Evaluation of flavonoids from Bauhinia megalandra leaves as inhibitors of glucose-6-phosphatase system, Phytother. Res. 19 (2005) 859–863. [15] G.S. Li, W.L. Jiang, X.D. Yue, G.W. Qu, J.W. Tian, J. Wu, F.H. Fu, Effect of astilbin on experimental diabetic nephropathy in vivo and in vitro, Planta. Med. 75 (2009) 1470–1475. [16] N. Mizushima, T. Yoshimori, How to interpret LC3 immunoblotting, Autophagy 3 (2007) 542–545. [17] G. Bjorkoy, T. Lamark, A. Brech, H. Outzen, M. Perander, A. Overvatn, H. Stenmark, T. Johansen, p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death, J. Cell Biol. 171 (2005) 603–614. [18] S. Pankiv, T.H. Clausen, T. Lamark, A. Brech, J.A. Bruun, H. Outzen, A. Overvatn, G. Bjorkoy, T. Johansen, p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy, J. Biol. Chem. 282 (2007) 24131–24145. [19] K. Tziomalos, V.G. Athyros, Diabetic nephropathy: new risk factors and improvements in diagnosis, Rev. Diabet. Stud. 12 (2015) 110–118. [20] M.J. Park, D.I. Kim, S.K. Lim, J.H. Choi, H.J. Han, K.C. Yoon, S.H. Park, High glucose-induced O-GlcNAcylated carbohydrate response element-binding protein (ChREBP) mediates mesangial cell lipogenesis and fibrosis: the possible role in the development of diabetic nephropathy, J. Biol. Chem. 289 (2014) 13519–13530. [21] J.S. Huang, C.T. Chuang, M.H. Liu, S.H. Lin, J.Y. Guh, L.Y. Chuang, Klotho attenuates high glucose-induced fibronectin and cell hypertrophy via the ERK1/2-p38 kinase signaling pathway in renal interstitial fibroblasts, Mol. Cell. Endocrinol. 390 (2014) 45–53. [22] L. Zhou, D.Y. Xu, W.G. Sha, L. Shen, G.Y. Lu, X. Yin, Long non-coding MIAT mediates high glucose-induced renal tubular epithelial injury, Biochem. Biophys. Res. Commun. 468 (2015) 726–732. [23] L. Zhou, D.Y. Xu, W.G. Sha, L. Shen, G.Y. Lu, X. Yin, M.J. Wang, High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway, J. Transl. Med. 13 (2015) 352. [24] Y. Ding, M.E. Choi, Autophagy in diabetic nephropathy, J. Endocrinol. 224 (2015) R15–R30. [25] T. Ma, J. Zhu, X. Chen, D. Zha, P.C. Singhal, G. Ding, High glucose induces autophagy in podocytes, Exp. Cell Res. 319 (2013) 779–789. [26] S. Periyasamy-Thandavan, M. Jiang, Q. Wei, R. Smith, X.M. Yin, Z. Dong, Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells, Kidney Int. 74 (2008) 631–640. [27] X. Zhao, G. Liu, H. Shen, B. Gao, X. Li, J. Fu, J. Zhou, Q. Ji, Liraglutide inhibits autophagy and apoptosis induced by high glucose through GLP-1R in renal tubular epithelial cells, Int. J. Mol. Med. 35 (2015) 684–692. [28] X.Q. Zhang, J.J. Dong, T. Cai, X. Shen, X.J. Zhou, L. Liao, High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells, Oncotarget 8 (2017) 24119–24129.
5. Conclusion In summary, this study demonstrated that HG induced cell apoptosis and autophagy in HK-2 cells. Treatment with astilbin reversed the induction of HG on cell apoptosis and autophagy. Astilbin attenuated HGinduced inactivation of the PI3K/Akt pathway. The inhibitor of Akt reversed the effect of astilbin on cell apoptosis and autophagy. The results provided new sight that astilbin might be served as a therapy agent for DN.
Conflict of interest statement No conflict of interests. 1180
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activity of curcumin by modulation of apoptosis and autophagy in human lung cancer A549 cells through inhibiting PI3K/Akt/mTOR pathway, Oncol. Rep. 39 (2018) 1523–1531. [34] G. Huang, J. Lv, T. Li, G. Huai, X. Li, S. Xiang, L. Wang, Z. Qin, J. Pang, B. Zou, Y. Wang, Notoginsenoside R1 ameliorates podocyte injury in rats with diabetic nephropathy by activating the PI3K/Akt signaling pathway, Int. J. Mol. Med. 38 (2016) 1179–1189. [35] Y. Ma, F. Chen, S. Yang, Y. Duan, Z. Sun, J. Shi, Silencing of TRB3 ameliorates diabetic tubule interstitial nephropathy via PI3K/AKT signaling in rats, Med. Sci. Monit. 23 (2017) 2816–2824. [36] C. Huang, M.Z. Lin, D. Cheng, F. Braet, C.A. Pollock, X.M. Chen, KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways, Sci. Rep. 6 (2016) 23884.
[29] Y. Wei, J. Gao, L. Qin, Y. Xu, H. Shi, L. Qu, Y. Liu, T. Xu, T. Liu, Curcumin suppresses AGEs induced apoptosis in tubular epithelial cells via protective autophagy, Exp. Ther. Med. 14 (2017) 6052–6058. [30] Z.H. Ouyang, W.J. Wang, Y.G. Yan, B. Wang, G.H. Lv, The PI3K/Akt pathway: a critical player in intervertebral disc degeneration, Oncotarget 8 (2017) 57870–57881. [31] D. Heras-Sandoval, J.M. Perez-Rojas, J. Hernandez-Damian, J. Pedraza-Chaverri, The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration, Cell Signal. 26 (2014) 2694–2701. [32] Z. Li, H. Dong, M. Li, Y. Wu, Y. Liu, Y. Zhao, X. Chen, M. Ma, Honokiol induces autophagy and apoptosis of osteosarcoma through PI3K/Akt/mTOR signaling pathway, Mol. Med. Rep. 17 (2018) 2719–2723. [33] F. Liu, S. Gao, Y. Yang, X. Zhao, Y. Fan, W. Ma, D. Yang, A. Yang, Y. Yu, Antitumor
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Astilbin inhibits high glucose-induced autophagy and apoptosis through the PI3K/Akt pathway in human proximal tubular epithelial cells Fang Chen
⁎,1
T
, Zhiqiang Sun1, Xiaoguang Zhu, Yali Ma
Department of Nephrology, Huaihe Hospital of Henan University, Kaifeng 475000, Henan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Diabetic nephropathy (DN) Astilbin Autophagy Cell apoptosis PI3K/Akt pathway
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. It has been found that astilbin, a flavonoid compound, exerts a protective effect on DN. However, the role of astilbin in autophagy during DN is unknown. The human proximal tubular epithelial cells (HK-2 cells) were treated with high glucose (HG, 30 mM) in the presence or absence of astilbin. Cell viability was measured by MTT assay. The autophagy was determined by detecting the expression of LC3-II and p62 using western blot. The cell apoptosis was evaluated by detecting the apoptosis rate, caspase-3 activity, and the expression of Bcl-2 and Bax. The expression levels of protein kinase B (Akt) and p-Akt were detected by western blot. To determine whether the phosphatidylinositol-3-kinase (PI3K)/Akt pathway was involved in the effect of astilbin, cells were treated with the inhibitor of Akt, LY294002. We found that astilbin (10 and 20 μM) did not affect the viability of HK-2 cells, but attenuated HG-induced cell viability. Astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells. The expression of p-Akt was inhibited by HG treatment, while the inhibitory effect of HG was attenuated by astilbin. Inhibition of the PI3K/ Akt signaling resisted the effect of astilbin on HG-induced apoptosis and autophagy. In conclusion, astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells through the PI3K/Akt pathway. The results indicated that astilbin might be a new therapeutic agent and be useful for improving clinical management of DN.
1. Introduction Diabetic nephropathy (DN) is a devastating complication of diabetes mellitus and accounts for a significant increase in morbidity and mortality in patients with diabetes [1]. It is well known that DN is the leading cause of end-stage renal disease and is becoming a serious health problem worldwide [1]. Currently, the therapies for DN focus on controlling blood glucose levels and blood pressure, and regulating the renin-angiotensin system (RAS) to inhibit the development and progression of DN [2,3]. Therefore, it is critically needed to further understand the pathogenesis of DN to identify new therapies for DN. Autophagy is a part of the catabolic process that degrades the damaged proteins and organelles, maintaining cell homeostasis under various stress conditions [4,5]. Accumulating evidence suggests that dysregulated autophagy plays pathogenic roles in various diseases [6]. The functional roles of autophagy in the kidney under normal and disease conditions have been gradually clarified. Autophagy protects kidney from induction of aging [7], hypoxia [8], and anticancer drugs [9,10]. Therefore, regulating autophagy activity in DN may be usually
for the management of DN. Astilbin (Fig. 1A) is a flavonoid compound and possesses a variety of activities, such as anti-inflammatory and immunoregulatory effects [11,12]. Diao et al. [13] reported that astilbin protected diabetic rat heart against ischemia-reperfusion (I/R) injury via inhibiting the HMGB1-dependent NF-κB signaling pathway. Astilbin also exhibited anti-hyperglycaemic effect by inhibiting the intact microsomal glucose6-phosphatase [14]. Moreover, astilbin also exerted a protective effect on DN [15], suggesting astilbin might be a potential agent in DN therapy. However, the role of astilbin in autophagy during DN is unknown. In this study, we investigated the effect of astilbin on autophagy and apoptosis in high glucose (HG)-induced human proximal tubular epithelial cells (HK-2 cells). We found that astilbin attenuated HG-induced autophagy and apoptosis in HK-2 cells.
Abbreviations: AKT, protein kinase B; DN, diabetic nephropathy; HG, high glucose; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol-3-kinase ⁎ Corresponding author at: Department of Nephrology, Huaihe Hospital of Henan University, No. 115 Ximen Street, Kaifeng 475000, Henan, PR China. E-mail address: [email protected] (F. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2018.07.072 Received 13 April 2018; Received in revised form 7 July 2018; Accepted 13 July 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Effect of astilbin on cell viability in HG-induced cells. (A) Chemical structure of astilbin. (B) Cell viability of HK-2 cells treated with different concentrations of glucose (5.5, 10, 20, and 30 mM). *P < 0.05 vs. cells without glucose treatment. (C and D) Effect of astilbin (10 and 20 μg/ml) on the viability of HK-2 cells treated with low concentration of glucose (5.5 mM) and high concentration of glucose (HG, 30 mM). *P < 0.05 vs. cells without treatment of glucose or astilbin. # P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
Fig. 2. Effect of astilbin on HG-induced autophagy in HK-2 cells. HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. The expression levels of LC3-II and p62 were detected by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
2. Materials and methods
2.2. MTT assay
2.1. Cell culture
HK-2 cells (1 × 104cells/well) were seeded in 96-well plates, and cultured for 24 h. Then 0.5 mg/ml of MTT solution was added to each well and incubated for another 4 h at 37 °C. Subsequently, the formazan crystals were dissolved by adding DMSO solution. Finally, the absorbance was measured at the wavelength of 570 nm using a Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).
HK-2 cells were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China). The cells were maintained in the DF12 medium (Gibco Laboratories, NY, USA) containing 10% FBS and cultured in a humidified incubator at 37 °C with 5% CO2. HK-2 cells were treated with appointed concentrations of HG, astilbin (Shanghai Tauto Biotech Co., Ltd., Shanghai, China; purity > 98%), or/and LY294002 (the inhibitor of Akt; Sigma-Aldrich, St. Louis, MO, USA) for 24 h.
2.3. Western blot HK-2 cell extracts were obtained using lysis buffer (Invitrogen, Carlsbad, CA, USA). Proteins in the cell extracts were separated by 12% SDS-PAGE gels. The concentration of the protein was measured by 1176
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Fig. 3. Effect of astilbin on HG-induced apoptosis of HK-2 cells. HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. (A) The apoptosis rate was detected by flow cytometry. (B) Detection of caspase-3 activity. (C) The expression levels of Bcl-2 and Bax were determined by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
2.4. Flow cytometry
using a BCA protein assay kit (Pierce Biotechnology, IL, USA). The proteins were transferred to nitrocellulose membranes and the membranes were blocked with 5% non-fat milk in TBST buffer at 37 °C for 1 h. The membranes were then incubated with primary antibodies against LC3, p62, Bcl-2, Bax, Akt, p-Akt, and β-actin (dilution 1: 500, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then the proteins were probed by incubating with HRP-conjugated secondary antibody (dilution 1: 2000, Abcam) for 2 h at 37 °C. Finally, the brands were detected with ECL reagent (Millipore, Billerica, MA, USA).
The treated HK-2 cells were harvested and stained using a Annexin V-PI Apoptosis Detection Kit (Abcam). After incubation for 30 min in the dark, the cell apoptosis ratio was measured using FACScan flow cytometer (Becton Dickenson, San Jose, CA, USA). 2.5. Detection of caspase-3 activity HK-2 cell extracts containing 50 μg of proteins were incubated with 1177
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Fig. 4. Effect of astilbin on the PI3K/Akt pathway under HG condition. To evaluate whether the PI3K/Akt pathway was affected by astilbin under HG condition, HK-2 cells were treated with HG in the presence or absence of astilbin (10 and 20 μg/ml) for 24 h. The expression levels of p-Akt and Akt were measured by western blot. *P < 0.05 vs. cells without treatment of glucose or astilbin. #P < 0.05 vs. cells treated with HG alone. Significance was determined by one-way ANOVA. Glu: glucose.
Fig. 5. Effect of LY294002 on astilbin-induced autophagy inhibition under HG condition. To evaluate whether the PI3K/Akt pathway was involved in the effect of astilbin on cell autophagy under HG condition, HK-2 cells were treated with HG (30 mM) in the presence or absence of astilbin (20 μg/ml) or the inhibitor of Akt (LY294002, 10 μM) for 24 h. The expression levels of LC3-II and p62 were detected by western blot. *P < 0.05 vs. cells treated with HG alone. #P < 0.05 vs. cells treated with HG and astilbin. Significance was determined by one-way ANOVA. Glu: glucose. LY: LY294002.
100 μM of the enzyme-specific colorimetric substrate Ac-DEVD-pNA at 37 °C for 2 h. The colorimetric release of p-nitroaniline was determined by detecting the absorbance at the wavelength of 405 nm.
3.2. Astilbin inhibited HG-induced autophagy in HK-2 cells The amount of LC3-II, the LC3-II/LC3-I ratio or LC3-II/(LC3I + LC3-II) ratio is now widely used to monitor autophagy. Because LC3-II exhibits more sensitive than LC3-I detected using western blot, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3II, may not be appropriate, and rather, comparison of the LC3-II amount among different groups is likely to be a more accurate method to reflect the autophagy [16]. In the current study, the expression level of LC3-II was detected by western blot. As shown in Fig. 2, we found that HG induced LC3-II expression, and astilbin treatment (10 and 20 μg/ml) attenuated the induction. The nucleoporin p62 (p62) is a protein complex that is down-regulated during autophagy and is widely used to monitor autophagic activity [17,18]. In Fig. 2, HG inhibited p62 expression, while astilbin treatment (10 and 20 μg/ml) attenuated the inhibition. The results indicated that astilbin attenuated HG-induced autophagy in HK-2 cells.
2.6. Statistical analysis Data are shown as mean ± SD of three independent experiments. Comparison was assessed by one-way analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Chicago, IL, USA). A difference was considered significant when p value was less than 0.05.
3. Results 3.1. Astilbin inhibited the viability of HK-2 cells under HG condition To determine the effect of glucose on cell viability, HK-2 cells were cultured with different concentrations of glucose (5.5, 10, 20, and 30 mM). The results in Fig. 1B showed that high concentration of glucose (30 mM) induced the viability of HK-2 cells, while the low concentrations of glucose (5.5, 10, and 20 mM) did not affect the cell viability. To evaluate the effect of astilbin on HG-induced viability, cells were cultured with 5.5 or 30 mM of glucose in the presence or absence of 10 or 20 μg/ml astilbin for 24 h. The results in Fig. 1C and D indicated that astilbin attenuated the effect of HG (30 mM) on cell viability, however, astilbin did not affect the viability of cells treated with low concentration of glucose (5.5 mM).
3.3. Astilbin inhibited HG-induced apoptosis in HK-2 cells To further investigate the role of astilbin in HG-induced cell apoptosis, the apoptosis rate was detected by flow cytometry. The apoptosis rate was increased in cells treated with HG, and astilbin decreased the apoptosis rate under HG condition (Fig. 3A). The caspase-3 activity was also measured. The results in Fig. 3B showed that HG increased caspase3 activity, but astilbin treatment decreased the caspase-3 activity compared to cells treated with HG. The expression levels of Bcl-2 and Bax were determined by western blot. The Bcl-2 expression was 1178
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Fig. 6. Effect of LY294002 on astilbin-induced apoptosis inhibition under HG condition. To determine whether the PI3K/Akt pathway was involved in the effect of astilbin on cell apoptosis under HG condition, HK-2 cells were treated with HG (30 mM) in the presence or absence of astilbin (20 μg/ml) or the inhibitor of Akt (LY294002, 10 μM) for 24 h. (A) The apoptosis rate was detected by flow cytometry. (B) Detection of caspase-3 activity. (C) The expression levels of Bcl-2 and Bax were determined by western blot. *P < 0.05 vs. cells treated with HG alone. #P < 0.05 vs. cells treated with HG and astilbin. Significance was determined by oneway ANOVA. Glu: glucose. LY: LY294002.
3.6. The PI3K/Akt signaling was involved in the role of astilbin on HGinduced apoptosis
decreased and Bax expression was increased in the cells treated with HG, however, when the cells were treated with astilbin, the Bcl-2 expression was increased and Bax expression was decreased compared with cells only treated with HG (Fig. 3C). The results suggested that astilbin inhibited HG-induced apoptosis of HK-2 cells.
To determine whether the PI3K/Akt pathway was involved in the effect of astilbin on cell apoptosis, cells were incubated with LY294002 and the cell apoptosis was measured. As shown in Fig. 6A and B, the apoptosis rate and caspase-3 activity were decreased after treatment with astilbin under HG condition, but LY294002 reversed the effects of astilbin on the apoptosis rate and caspase-3 activity. The expression level of Bcl-2 was increased and the expression level of Bax was decreased in cells treated with astilbin under HG condition, but LY294002 reversed the effects of astilbin on Bcl-2 and Bax expression (Fig. 6C). The results indicated that the PI3K/Akt signaling was involved in the effect of astilbin on HG-induced cell apoptosis.
3.4. Astilbin activated the PI3K/Akt pathway under HG condition To evaluate whether the PI3K/Akt pathway was affected by astilbin under HG condition, the expression levels of p-Akt and Akt were measured by western blot. As shown in Fig. 4, expression of p-Akt was inhibited by HG treatment, while the inhibitory effect of HG was attenuated by astilbin. The treatment with HG and/or astilbin showed no significant effect on the expression of Akt, indicating that astilbin activated the PI3K/Akt pathway under HG condition.
4. Discussion 3.5. The PI3K/Akt signaling was involved in the role of astilbin on HGinduced autophagy
Elevated glucose level is one of the main risk factors for the development and progression of DN [19]. High blood sugar may lead to the formation of advanced glycation end products, which induce inflammation in the kidney and promote the development of DN [19]. HG is usually used for the establishment of in vitro model of DN [20,21]. Previous studies indicated that treatment with HG showed significant reduction in cell viability of HK-2 cells and induced renal tubular epithelial injury [22,23]. In our work, we found that HG (30 mM) inhibited the viability of HK-2 cells, while the low concentrations of glucose (5.5, 10, and 20 mM) did not affect the cell viability.
To evaluate whether the PI3K/Akt pathway was involved in the effect of astilbin on autophagy, cells were incubated with the inhibitor of Akt (LY294002) and the autophagy level was measured. As shown in Fig. 5, astilbin reduced the LC3-II/LC3-I ratio and increased p62 expression level under HG condition. However, LY294002 reversed astilbin-induced inhibitory effect on autophagy under HG condition. The results suggested that the PI3K/Akt signaling was involved in the effect of astilbin on HG-induced autophagy. 1179
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References
Autophagy plays a critical role in degrading cellular protein aggregates and damaged organelles, promoting cell survival and maintaining homeostasis. However, excessive autophagy can also contribute to apoptosis. Thus, autophagy may be deleterious. [24]. It was reported that within 24 h of exposure to HG condition induced autophagy in podocytes, suggesting that the acute exposure to HG could induce autophagy [25]. Administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys, and in cultured renal proximal tubular cells, cisplatin induced autophagy and apoptosis [26]. In the present study, we found that HG induced LC3-II expression, inhibited p62 expression, and increased the apoptosis rate in HK-2 cells, suggesting that HG induced autophagy and apoptosis in HK-2 cells. However, astilbin attenuated the induction effect of HG on autophagy and apoptosis in HK-2 cells. Zhao et al. [27] reported that liraglutide suppressed HG-induced autophagy and apoptosis through glucagon-like peptide-1 receptor in renal tubular epithelial cells. However, some studies displayed different results. A recent study showed that HG induced apoptosis via up-regulation of Bim expression, but not brought a significant change in autophagy in HK-2 cells. Knockdown of Bim inhibited HG-induced apoptosis and also sensitized HK-2 cells to autophagy under HG condition [28]. Curcumin reversed advanced glycation or glycoxidation end-product (AGE)-induced apoptosis by promoting autophagy through the PI3K/Akt signaling pathway in NRK-52E rat renal tubular epithelial cells [29]. These inconsistent findings may result from the different cell lines used. The PI3K/Akt pathway is a main intracellular signaling pathway that regulates multiple biological processes, such as cell proliferation, differentiation, and apoptosis under the physiological and pathological conditions [30]. It has been demonstrated that the PI3K/Akt pathway is closely related to autophagy [31]. Recently, Li et al. [32] reported that honokiol, the main active constituent of Magnolia officinalis, induced autophagy and apoptosis of osteosarcoma cells through the PI3K/Akt/ mammalian target of rapamycin (mTOR) signaling pathway. Curcumin exhibited the antitumor effect via regulating cell apoptosis and autophagy in human lung cancer A549 cells through inhibiting the PI3K/Akt/ mTOR pathway [33]. These studies revealed the important role of the PI3K/Akt pathway in regulating cell apoptosis and autophagy. The PI3K/Akt pathway was also reported to play an important role in DN. Huang et al. reported that Notoginsenoside R1 treatment ameliorates podocyte injury in rats with DN through suppressing inflammation and apoptosis via the PI3K/Akt signaling pathway [34]. Silencing of TRB3 ameliorated diabetic tubule interstitial nephropathy via the PI3K/AKT signaling in diabetic rats, suggesting that silencing of TRB3 had a significant ameliorative effect on diabetic nephropathy in rats [35]. KCa3.1 knockdown alleviated dysfunctional tubular autophagy in diabetic nephropathy through the PI3K/Akt/mTOR signaling pathway [36]. In the present study, we also found that the PI3K/Akt signaling was involved in the role of astilbin on HG-induced autophagy and cell apoptosis. However, the findings were based on the in vitro study, an in vivo study is still needed.
[1] F.B. Hu, Globalization of diabetes: the role of diet, lifestyle, and genes, Diabetes Care 34 (2011) 1249–1257. [2] P. Ruggenenti, P. Cravedi, G. Remuzzi, The RAAS in the pathogenesis and treatment of diabetic nephropathy, Nat. Rev. Nephrol. 6 (2010) 319–330. [3] N.A. Calcutt, M.E. Cooper, T.S. Kern, A.M. Schmidt, Therapies for hyperglycaemiainduced diabetic complications: from animal models to clinical trials, Nat. Rev. Drug Discov. 8 (2009) 417–429. [4] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through cellular self-digestion, Nature 451 (2008) 1069–1075. [5] J.H. Hurley, B.A. Schulman, Atomistic autophagy: the structures of cellular selfdigestion, Cell 157 (2014) 300–311. [6] W. Xie, J. Zhou, Aberrant regulation of autophagy in mammalian diseases, Biol. Lett. 14 (2018). [7] B. Hartleben, M. Godel, C. Meyer-Schwesinger, S. Liu, T. Ulrich, S. Kobler, T. Wiech, F. Grahammer, S.J. Arnold, M.T. Lindenmeyer, C.D. Cohen, H. Pavenstadt, D. Kerjaschki, N. Mizushima, A.S. Shaw, G. Walz, T.B. Huber, Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice, J. Clin. Invest. 120 (2010) 1084–1096. [8] T. Kimura, Y. Takabatake, A. Takahashi, J.Y. Kaimori, I. Matsui, T. Namba, H. Kitamura, F. Niimura, T. Matsusaka, T. Soga, H. Rakugi, Y. Isaka, Autophagy protects the proximal tubule from degeneration and acute ischemic injury, J. Am. Soc. Nephrol. 22 (2011) 902–913. [9] C. Yang, V. Kaushal, S.V. Shah, G.P. Kaushal, Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells, Am. J. Physiol. Ren. Physiol. 294 (2008) F777–787. [10] G.P. Kaushal, V. Kaushal, C. Herzog, C. Yang, Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity, Autophagy 4 (2008) 710–712. [11] J. Yu, Z. Xiao, R. Zhao, C. Lu, Y. Zhang, Astilbin emulsion improves guinea pig lesions in a psoriasis-like model by suppressing IL-6 and IL-22 via p38 MAPK, Mol. Med. Rep. 17 (2017) 3789–3796. [12] Q.F. Meng, Z. Zhang, Y.J. Wang, W. Chen, F.F. Li, L.T. Yue, C.J. Zhang, H. Li, M. Zhang, C.C. Wang, P. Zhang, H. Chen, R.S. Duan, S.M. Sun, Y.B. Li, Astilbin ameliorates experimental autoimmune myasthenia gravis by decreased Th17 cytokines and up-regulated T regulatory cells, J. Neuroimmunol. 298 (2016) 138–145. [13] H. Diao, Z. Kang, F. Han, W. Jiang, Astilbin protects diabetic rat heart against ischemia-reperfusion injury via blockade of HMGB1-dependent NF-kappaB signaling pathway, Food Chem. Toxicol. 63 (2014) 104–110. [14] O. Estrada, M. Hasegawa, F. Gonzalez-Mujica, N. Motta, E. Perdomo, A. Solorzano, J. Mendez, B. Mendez, E.G. Zea, Evaluation of flavonoids from Bauhinia megalandra leaves as inhibitors of glucose-6-phosphatase system, Phytother. Res. 19 (2005) 859–863. [15] G.S. Li, W.L. Jiang, X.D. Yue, G.W. Qu, J.W. Tian, J. Wu, F.H. Fu, Effect of astilbin on experimental diabetic nephropathy in vivo and in vitro, Planta. Med. 75 (2009) 1470–1475. [16] N. Mizushima, T. Yoshimori, How to interpret LC3 immunoblotting, Autophagy 3 (2007) 542–545. [17] G. Bjorkoy, T. Lamark, A. Brech, H. Outzen, M. Perander, A. Overvatn, H. Stenmark, T. Johansen, p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death, J. Cell Biol. 171 (2005) 603–614. [18] S. Pankiv, T.H. Clausen, T. Lamark, A. Brech, J.A. Bruun, H. Outzen, A. Overvatn, G. Bjorkoy, T. Johansen, p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy, J. Biol. Chem. 282 (2007) 24131–24145. [19] K. Tziomalos, V.G. Athyros, Diabetic nephropathy: new risk factors and improvements in diagnosis, Rev. Diabet. Stud. 12 (2015) 110–118. [20] M.J. Park, D.I. Kim, S.K. Lim, J.H. Choi, H.J. Han, K.C. Yoon, S.H. Park, High glucose-induced O-GlcNAcylated carbohydrate response element-binding protein (ChREBP) mediates mesangial cell lipogenesis and fibrosis: the possible role in the development of diabetic nephropathy, J. Biol. Chem. 289 (2014) 13519–13530. [21] J.S. Huang, C.T. Chuang, M.H. Liu, S.H. Lin, J.Y. Guh, L.Y. Chuang, Klotho attenuates high glucose-induced fibronectin and cell hypertrophy via the ERK1/2-p38 kinase signaling pathway in renal interstitial fibroblasts, Mol. Cell. Endocrinol. 390 (2014) 45–53. [22] L. Zhou, D.Y. Xu, W.G. Sha, L. Shen, G.Y. Lu, X. Yin, Long non-coding MIAT mediates high glucose-induced renal tubular epithelial injury, Biochem. Biophys. Res. Commun. 468 (2015) 726–732. [23] L. Zhou, D.Y. Xu, W.G. Sha, L. Shen, G.Y. Lu, X. Yin, M.J. Wang, High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway, J. Transl. Med. 13 (2015) 352. [24] Y. Ding, M.E. Choi, Autophagy in diabetic nephropathy, J. Endocrinol. 224 (2015) R15–R30. [25] T. Ma, J. Zhu, X. Chen, D. Zha, P.C. Singhal, G. Ding, High glucose induces autophagy in podocytes, Exp. Cell Res. 319 (2013) 779–789. [26] S. Periyasamy-Thandavan, M. Jiang, Q. Wei, R. Smith, X.M. Yin, Z. Dong, Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells, Kidney Int. 74 (2008) 631–640. [27] X. Zhao, G. Liu, H. Shen, B. Gao, X. Li, J. Fu, J. Zhou, Q. Ji, Liraglutide inhibits autophagy and apoptosis induced by high glucose through GLP-1R in renal tubular epithelial cells, Int. J. Mol. Med. 35 (2015) 684–692. [28] X.Q. Zhang, J.J. Dong, T. Cai, X. Shen, X.J. Zhou, L. Liao, High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells, Oncotarget 8 (2017) 24119–24129.
5. Conclusion In summary, this study demonstrated that HG induced cell apoptosis and autophagy in HK-2 cells. Treatment with astilbin reversed the induction of HG on cell apoptosis and autophagy. Astilbin attenuated HGinduced inactivation of the PI3K/Akt pathway. The inhibitor of Akt reversed the effect of astilbin on cell apoptosis and autophagy. The results provided new sight that astilbin might be served as a therapy agent for DN.
Conflict of interest statement No conflict of interests. 1180
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F. Chen et al.
activity of curcumin by modulation of apoptosis and autophagy in human lung cancer A549 cells through inhibiting PI3K/Akt/mTOR pathway, Oncol. Rep. 39 (2018) 1523–1531. [34] G. Huang, J. Lv, T. Li, G. Huai, X. Li, S. Xiang, L. Wang, Z. Qin, J. Pang, B. Zou, Y. Wang, Notoginsenoside R1 ameliorates podocyte injury in rats with diabetic nephropathy by activating the PI3K/Akt signaling pathway, Int. J. Mol. Med. 38 (2016) 1179–1189. [35] Y. Ma, F. Chen, S. Yang, Y. Duan, Z. Sun, J. Shi, Silencing of TRB3 ameliorates diabetic tubule interstitial nephropathy via PI3K/AKT signaling in rats, Med. Sci. Monit. 23 (2017) 2816–2824. [36] C. Huang, M.Z. Lin, D. Cheng, F. Braet, C.A. Pollock, X.M. Chen, KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways, Sci. Rep. 6 (2016) 23884.
[29] Y. Wei, J. Gao, L. Qin, Y. Xu, H. Shi, L. Qu, Y. Liu, T. Xu, T. Liu, Curcumin suppresses AGEs induced apoptosis in tubular epithelial cells via protective autophagy, Exp. Ther. Med. 14 (2017) 6052–6058. [30] Z.H. Ouyang, W.J. Wang, Y.G. Yan, B. Wang, G.H. Lv, The PI3K/Akt pathway: a critical player in intervertebral disc degeneration, Oncotarget 8 (2017) 57870–57881. [31] D. Heras-Sandoval, J.M. Perez-Rojas, J. Hernandez-Damian, J. Pedraza-Chaverri, The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration, Cell Signal. 26 (2014) 2694–2701. [32] Z. Li, H. Dong, M. Li, Y. Wu, Y. Liu, Y. Zhao, X. Chen, M. Ma, Honokiol induces autophagy and apoptosis of osteosarcoma through PI3K/Akt/mTOR signaling pathway, Mol. Med. Rep. 17 (2018) 2719–2723. [33] F. Liu, S. Gao, Y. Yang, X. Zhao, Y. Fan, W. Ma, D. Yang, A. Yang, Y. Yu, Antitumor
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