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Dihydroartemisinin inhibits the proliferation of IgAN mesangial cells through the mTOR signaling pathway
International Immunopharmacology 80 (2020) 106125
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Preliminary report
Dihydroartemisinin inhibits the proliferation of IgAN mesangial cells through the mTOR signaling pathway Ming Xia, Di Liu, Xiaofang Tang, Yexin Liu, Haiyang Liu, Yu Liu, Guochun Chen, Hong Liu
T ⁎
Department of Nephrology, The Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Kidney Disease and Blood Purification, No. 139 Renmin Middle Rd, Changsha 410011, Hunan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: IgA nephropathy Dihydroartemisinin rapamycin/ribosomal protein S6 kinase beta-1 pathway Mesangial cell proliferation
IgA nephropathy (IgAN) is an autoimmune kidney disease and is the most prevalent form of glomerular kidney disease in China and worldwide. IgA immune complex deposition accompanied by mesangial cell proliferation and mesangial matrix expansion is the most basic pathological feature of IgAN. Dihydroartemisinin (DHA), an antimalarial drug, was recently reported to be effective in treating autoimmune diseases. However, its potential therapeutic role in IgAN is relatively unstudied. The aim of this study was to investigate the pharmacological effects and the underlying mechanisms of DHA in the treatment of IgAN. In this study, renal biopsy specimens were collected for immunohistochemistry. In vitro, 25 μg/ml concentrations of aggregated IgA1 (aIgA1) was used to construct the IgAN mesangial cell model. Stimulated human mesangial cells (HMCs) were treated for 24 h with DHA (0–15 μM) and were collected for western blot analyses. Cell proliferation was assessed by Cell Counting Kit 8 (CCK8) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. In vitro, our results showed that DHA could downregulate the mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 (mTOR/S6K1) signaling pathway, promote cell autophagy, and ameliorate cell proliferation in aIgA1-induced HMCs. The results suggested that DHA may represent a novel class of mTOR inhibitor and promote an anti-proliferation effect in IgAN HMCs, which provides an alternative approach for IgAN treatment.
1. Introduction IgA nephropathy (IgAN) is the most prevalent form of glomerular kidney disease worldwide, particularly in Asian areas, and has a significant morbidity and mortality rate, with 30 to 40% of patients having impaired renal function and eventually developing end-stage renal disease (ESRD) after 20–30 years [1,2]. IgAN is characterized by immune complex deposition in the mesangial areas accompanied by mesangial cell proliferation. Mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 (mTOR/S6K1), an important cell growth coordination pathway, functions as a negative controller of autophagy which can attenuate kidney injury of IgAN [3,4]. Data revealed that the activation of the mTOR signal is a critical event to induce and accelerate the progression of IgAN [5–8]. Dihydroartemisinin (DHA) is an active metabolite compound derived from artemisinin and is widely used as an antimalarial therapeutic agent [9]. Much evidence has emerged to elucidate the biological effects of DHA, especially in immunomodulation and cell proliferation [10,11]. Based on the fact that IgAN is an autoimmune disease, glomerular mesangial cell proliferation is the most basic pathological
⁎
characteristic and is related to IgAN progression [12,13]. Thus, it is imperative to determine whether DHA has a therapeutic effect on IgAN. Here, we investigated the potential role of DHA therapy and for the first time, described the mechanism of DHA regarding the mTOR/S6K1 pathway in IgAN. Our results showed that DHA could suppress mTOR/ S6K1 signaling, induce autophagy, and inhibit proliferation of IgAN human mesangial cells (HMCs) stimulated by aggregated IgA1 (aIgA1). 2. Materials and methods 2.1. Cell culture and treatment The human glomerular mesangial cell line (CBR130735, Cellbio, China) was cultured in antibiotic-free Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Monomeric human IgA1 (Abcam) was heated and aggregated at 65℃ for 150 min on a dry plate heater to obtain aIgA1 as previously described [14]. DHA (purity > 98% by high-performance liquid chromatography) was purchased from ApexBio Technology (Houston, US), and dissolved in dimethyl sulfoxide
Corresponding author. E-mail address: [email protected] (H. Liu).
https://doi.org/10.1016/j.intimp.2019.106125 Received 30 July 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Fig. 1. Upregulation of pmTOR and pS6K1 in the kidneys of IgAN patients. (A) Representative images of glomerular immunohistostaining from IgAN patients and controls with pmTOR and pS6K1. (B) The average optical density of pmTOR and pS6K1 were analyzed by ImageJ software. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 specimens in each group).
MA), with periodic acid-Schiff (PAS) used instead of primary antibodies as negative controls for all staining. Finally, horseradish peroxidase (HRP)-conjugated polymer (Abcam, Cambridge, MA) was used for the visualized detection under light microscopy (Nikon Tokyo, Japan). OD values were analyzed by ImageJ software (Media Cybernetics, Inc., Bethesda, MD, USA). All the analyses were repeated no less than three times, and representative images are displayed. The use of renal specimens of IgAN patients was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University according to the Declaration of Helsinki. 2.4. Western blotting Total proteins were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) as previously described [16]. The lysate was centrifuged at 12,000g for 30 min at 4 °C. After centrifugation, supernatants were obtained for the evaluation of total protein concentration with bovine serum albumin (BSA) as a standard using a BCA protein assay kit (Pierce, Thermo). In brief, 30 μg of protein was used for electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% BSA at room temperature for 1 h, membranes were incubated overnight at 4 °C with the primary antibodies: pmTOR (Abcam, Cambridge, MA), pS6K1 (Abcam, Cambridge, MA), LC3B (GeneTex, Irvine, USA), and β-actin (Proteintech, Rosemont, USA). After washing with phosphate-buffered saline with Tween (PBST), blots were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG, Abcam, Cambridge, MA) at room temperature for 1 h, and enhanced chemiluminescence (Millipore) was used to visualize the bands.
Fig. 2. The construction of the IgAN HMCs model. HMCs were stimulated with different concentrations of aIgA1. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
(DMSO) to prepare a stock solution (20 mM) and stored at −20 °C. After starvation for 12 h in serum-free medium, cells were then incubated with aIgA1 alone or with a combination of DHA for 24 h. 2.2. Cell proliferation assay Cell proliferation was examined with a light microscope and accessed by Cell Counting Kit-8 (CCK8) assay (Dojindo, Kumamoto, Japan) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Servicebio, China). For CCK8, 10 μl of CCK8 was added to each well and incubated at 37 °C. Cell viability was determined by measuring optical density (OD) at a 450-nm wavelength. For MTT, 20 μl of MTT was added to each well and incubated at 37 °C. Subsequently, 200 μl of DMSO was added. The absorbance was detected at 490 nm using a spectrophotometer.
2.5. Statistics Statistical differences were analyzed by Student’s t-test, and p < 0.05 was considered statistically significant. Analyses were conducted with GraphPad Prism 6.0 statistical software.
2.3. Immunohistochemistry staining 3. Results Formaldehyde-fixed and paraffin-embedded tissue sections (4 mm thick) were used for immunohistochemistry as previously described [15]. After dehydration, slides in citrate solution were subjected to a microwave antigen retrieval process. Primary antibodies were employed against the phosphorylated mTOR (pmTOR) (Abcam, Cambridge, MA) and phosphorylated S6K1 (pS6K1) (Abcam, Cambridge,
3.1. pmTOR and pS6K1 were abnormally elevated in IgAN biopsy We had previously constructed an IgAN rat model and found that expressions of pmTOR and pS6K1 were upregulated. That is, the mTOR/S6K1 pathway was activated in IgAN. In addition, the activation 2
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Fig. 3. DHA inhibits HMC proliferation in the IgAN HMC model. (A) The proliferation of HMCs was assessed by CCK8 assay. (B) The proliferation rate of HMCs was measured by MTT assay. (C) Treatment of aIgA1-induced HMCs without or with different concentrations of DHA for 24 h. Representative light microscopy images are presented. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
concentration-dependent manner under 10 μM, while the OD values measured by the two methods were slightly different (Fig. 3A and 3B). In addition, light microscopy of HMCs stimulated with different concentrations of DHA is shown (Fig. 3C).
of this pathway was crucial for the proliferation of mesangial cells [4]. Here, pmTOR and pS6K1 were further detected by immunohistochemistry staining in the renal biopsy section of IgAN patients. Patients with minor glomerular lesions (MCD) were used as controls. The results showed elevated expression of pmTOR and pS6K1 in renal tissue specimens of IgAN compared to MCD (Fig. 1A). Using image analysis software, the average optical density of the pmTOR and pS6K1 staining were significantly higher in IgAN patients than in controls (Fig. 1B).
3.3. DHA suppressed mTOR/S6K1 signaling Increasing evidence indicates that mTOR is a central controller of proliferation and survival, we hypothesized that DHA might disrupt cell proliferation by suppressing mTOR/S6K1 signaling. We examined the effect of DHA on the mTOR signaling pathway in IgAN HMCs in vitro. By western blot analysis, we found that DHA suppressed phosphorylation of mTOR, and consistently inhibited phosphorylation of S6K1 in a dose-response experiment, especially with 15-μM DHA treatment. In addition, we also tested autophagy representative protein LC3B expression. DHA (above 5 μM) exhibited an obvious stimulatory effect on LC3B expression (Fig. 4).
3.2. DHA diminished proliferation of aIgA1-induced mesangial cells To investigate whether DHA can inhibit the proliferation of IgAN HMCs in vitro, we used different concentrations of aIgA1 to stimulate HMCs and found the most suitable concentration (25 μg/ml) to construct an IgAN HMC model (Fig. 2). Stimulated HMCs were treated with DHA for 24 h at concentrations of 0–15 μM. The MTT and CCK8 assay results showed that DHA diminished cell proliferation in a 3
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Fig. 4. DHA reduces phosphorylation of mTOR/S6K1 signaling in IgAN. Western blot analysis for pmTOR, pS6K1, and LC3B expressions in DHA-treated IgAN HMCs. Data are presented as mean ± SD. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
4. Discussion
protein in HMCs that received DHA. This indicated that DHA had antiinflammatory and autophagy promoting effects in IgAN [33]. However, this research was only a preliminary experiment, many questions could arise from the current work. First, mTOR functions at least two complexes, mTORC1 and mTORC2. Whether DHA affects both compounds or whether it affects the complex integrity warrants further investigation and validation. Second, further research is required to determine whether DHA directly regulates the upstream molecules of mTOR, for example, phosphatidylinositol 3 kinase (PI3K). Third, more results are needed to confirm the effects of cell proliferation and autophagy, such as cell cycle experiment and electron microscope observation of autophagy flow. Reports have shown that DHA could exhibit a significant anti-proliferation effect via transferrin receptor 1 (CD71) and the mTOR/S6K1 pathway in tumors [32,34,35]. Given that CD71 also plays a key role in trapping IgA1 immune complexes in situ and mesangial proliferation in IgAN [36], we believe that DHA may decrease mesangial cell proliferation by inhibiting mTOR/S6K1 pathway and affecting IgA1 immune complex-mediated activation of CD71 expression in IgA nephropathy, which may be a new mechanism beyond affecting autophagy. However, further study is still needed to explore the potential effects of DHA on IgAN. In conclusion, the present study supports a protective role of DHA in treating IgAN by inhibiting the mTOR/S6K1 pathway, enhancing autophagy, and ameliorating mesangial cell proliferation. This provides a new clue for IgAN treatment.
In this study, the IgAN HMCs model was well-established [14], and for the first time, we described the effects of DHA on mesangial cells. We demonstrated that DHA could prevent the proliferation of mesangial cells in IgAN and through inhibition of mTOR/S6K1. Our report suggests that DHA is not only a possible mTOR inhibitor, but also has great potential for IgAN therapy. The first description of IgA nephropathy was proposed by Jean Berger in 1968, and it is also named Berger’ s disease [17]. In IgAN, increasing mesangial matrix and hypercellularity are common pathological changes, making it crucial to find ways to control mesangial cell proliferation and slow its progression. DHA is derived from artemisinin, an effective constituent originally extracted from traditional Chinese medicine Artemisia annua L [18,19]. Although artemisinin displays abundant biology effects, it has attracted increasing attention in treating lupus in recent decades [20–24]. However, only studies have examined DHA in renal disease [25–28]. Thus, the possible role of DHA in nephropathy remains unclear. Increasing evidence demonstrates that DHA can potently inhibit cell proliferation, in particular, through the proteins regulated by mTOR, such as FAK, MMP-2, and NF-κB [29–31]. In addition, research has also shown that DHA could potently inhibit mTORC1-mediated pS6K1 in the tumor cells [32]. These led us to investigate whether DHA primarily inhibits mTOR signaling and then affects its downstream. In the previous study, we found that mTOR-mediated autophagy inhibition may result in mesangial cell proliferation in IgAN, while the inhibitor of mTOR could enhance repressed autophagy and attenuates aggressive progression in a rat model of IgAN [4]. Here, we hypothesized that DHA is able to affect IgAN through a similar mechanism. Our results showed a significant anti-proliferation effect on HMCs when treated with DHA (5–15 μM). In addition, our results also showed downregulation in phosphorylation of mTOR/S6K1 and upregulation of LC3B autophagy
CRediT authorship contribution statement Ming Xia: Investigation, Methodology, Writing - original draft. Di Liu: Software, Validation. Xiaofang Tang: Software, Formal analysis. Yexin Liu: Writing - review & editing. Haiyang Liu: Data curation. Yu Liu: Project administration, Funding acquisition. Guochun Chen: 4
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Conceptualization, Writing - review & editing. Hong Liu: Supervision, Funding acquisition.
[13] L. He, M.J. Livingston, Z. Dong, Autophagy in acute kidney injury and repair, Nephron. Clin. Pract. 127 (1–4) (2014) 56–60. [14] Y.Y. Hyun, et al., Adipose-derived stem cells improve renal function in a mouse model of IgA nephropathy, Cell Transplant. 21 (11) (2012) 2425–2439. [15] L.Y. He, et al., Synthetic double-stranded RNA Poly(I:C) aggravates IgA nephropathy by triggering IgA class switching recombination through the TLR3-BAFF Axis, Am. J. Nephrol. 42 (3) (2015) 185–197. [16] L.Y. He, et al., Regulation of IgA class switch recombination in immunoglobulin A nephropathy: retinoic acid signaling and BATF, Am. J. Nephrol. 43 (3) (2016) 179–194. [17] J. Berger, N. Hinglais, Intercapillary deposits of IgA-IgG, J. Urol. Nephrol (Paris) 74 (9) (1968) 694–695. [18] Z. Chang, The discovery of Qinghaosu (artemisinin) as an effective anti-malaria drug: A unique China story, Sci. China Life Sci. 59 (1) (2016) 81–88. [19] N.J. White, T.T. Hien, F.H. Nosten, A Brief History of Qinghaosu, Trends Parasitol. 31 (12) (2015) 607–610. [20] K. Kumari, et al., Transcriptome analysis of genes associated with breast cancer cell motility in response to Artemisinin treatment, BMC Cancer 17 (1) (2017) 858. [21] C.J. Paddon, et al., High-level semi-synthetic production of the potent antimalarial artemisinin, Nature 496 (7446) (2013) 528–532. [22] X. Mu, C. Wang, Artemisinins-a promising new treatment for systemic lupus erythematosus: a descriptive review, Curr. Rheumatol. Rep. 20 (9) (2018) 55. [23] X. Cui, et al., Traditional Chinese medicine and related active compounds against hepatitis B virus infection, Biosci. Trends 4 (2) (2010) 39–47. [24] T. Li, et al., Anti-inflammatory and immunomodulatory mechanisms of artemisinin on contact hypersensitivity, Int. Immunopharmacol. 12 (1) (2012) 144–150. [25] L. Li, et al., Dihydroartemisinin up-regulates VE-cadherin expression in human renal glomerular endothelial cells, J. Cell. Mol. Med. 22 (3) (2018) 2028–2032. [26] B. Zhang, et al., Dihydroartemisinin attenuates renal fibrosis through regulation of fibroblast proliferation and differentiation, Life Sci. 223 (2019) 29–37. [27] X. Liu, et al., Dihydroartemisinin attenuates lipopolysaccharide-induced acute kidney injury by inhibiting inflammation and oxidative stress, Biomed. Pharmacother. 117 (2019) 109070. [28] Z. Cheng, et al., Dihydroartemisinin ameliorates sepsis-induced hyperpermeability of glomerular endothelium via up-regulation of occludin expression, Biomed. Pharmacother. 99 (2018) 313–318. [29] B. Wu, et al., Dihydroartiminisin inhibits the growth and metastasis of epithelial ovarian cancer, Oncol. Rep. 27 (1) (2012) 101–108. [30] C.Z. Zhang, et al., Dihydroartemisinin exhibits antitumor activity toward hepatocellular carcinoma in vitro and in vivo, Biochem. Pharmacol. 83 (9) (2012) 1278–1289. [31] S.J. Wang, et al., Dihydroartemisinin inactivates NF-kappaB and potentiates the anti-tumor effect of gemcitabine on pancreatic cancer both in vitro and in vivo, Cancer Lett. 293 (1) (2010) 99–108. [32] Y. Odaka, et al., Dihydroartemisinin inhibits the mammalian target of rapamycinmediated signaling pathways in tumor cells, Carcinogenesis 35 (1) (2014) 192–200. [33] D.C. Rubinsztein, et al., In search of an “autophagomometer”, Autophagy 5 (5) (2009) 585–589. [34] H. Jin, et al., Dihydroartemisinin and gefitinib synergistically inhibit NSCLC cell growth and promote apoptosis via the Akt/mTOR/STAT3 pathway, Mol. Med. Rep. 16 (3) (2017) 3475–3481. [35] Q. Ba, et al., Dihydroartemisinin exerts its anticancer activity through depleting cellular iron via transferrin receptor-1, PLoS One 7 (8) (2012) e42703. [36] I.C. Moura, et al., Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy, J. Exp. Med. 194 (4) (2001) 417–425.
Acknowledgments We sincerely appreciate the time and effort of all who contributed to this study. Funding This work was supported by research grants (81770714), (81470947), and (81570622) from the National Natural Science Foundation of China. It was also funded by the postgraduate innovation project of Central South University in China (Project no. 2018zzts920). Declaration of Competing Interest No conflicts of interest, financial or otherwise, are declared by the authors. References [1] K.N. Lai, et al., IgA nephropathy, Nat. Rev. Dis. Primers 2 (2016) 16001. [2] S.J. Barbour, et al., Individuals of Pacific Asian origin with IgA nephropathy have an increased risk of progression to end-stage renal disease, Kidney Int. 84 (5) (2013) 1017–1024. [3] R.A. Saxton, D.M. Sabatini, mTOR signaling in growth, metabolism, and disease, Cell 168 (6) (2017) 960–976. [4] D. Liu, et al., Rapamycin enhances repressed autophagy and attenuates aggressive progression in a rat model of IgA nephropathy, Am. J. Nephrol. 45 (4) (2017) 293–300. [5] R.C. Monteiro, et al., Pathogenic significance of IgA receptor interactions in IgA nephropathy, Trends Mol. Med. 8 (10) (2002) 464–468. [6] H. Tamouza, et al., Transferrin receptor engagement by polymeric IgA1 induces receptor expression and mesangial cell proliferation: role in IgA nephropathy, Contrib. Nephrol. 157 (2007) 144–147. [7] J. Tian, et al., The Akt/mTOR/p70S6K pathway is activated in IgA nephropathy and rapamycin may represent a viable treatment option, Exp. Mol. Pathol. 99 (3) (2015) 435–440. [8] H. Tamouza, et al., The IgA1 immune complex-mediated activation of the MAPK/ ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy, Kidney Int. 82 (12) (2012) 1284–1296. [9] Y. Li, Qinghaosu (artemisinin): chemistry and pharmacology, Acta Pharmacol. Sin. 33 (9) (2012) 1141–1146. [10] L. Bai, et al., Immunosuppressive effect of artemisinin and hydroxychloroquine combination therapy on IgA nephropathy via regulating the differentiation of CD4+ T cell subsets in rats, Int. Immunopharmacol. 70 (2019) 313–323. [11] R. Lin, et al., Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells, Cancer Lett. 381 (1) (2016) 165–175. [12] J. Novak, et al., Glycosylation of IgA1 and pathogenesis of IgA nephropathy, Semin. Immunopathol. 34 (3) (2012) 365–382.
5
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Preliminary report
Dihydroartemisinin inhibits the proliferation of IgAN mesangial cells through the mTOR signaling pathway Ming Xia, Di Liu, Xiaofang Tang, Yexin Liu, Haiyang Liu, Yu Liu, Guochun Chen, Hong Liu
T ⁎
Department of Nephrology, The Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Kidney Disease and Blood Purification, No. 139 Renmin Middle Rd, Changsha 410011, Hunan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: IgA nephropathy Dihydroartemisinin rapamycin/ribosomal protein S6 kinase beta-1 pathway Mesangial cell proliferation
IgA nephropathy (IgAN) is an autoimmune kidney disease and is the most prevalent form of glomerular kidney disease in China and worldwide. IgA immune complex deposition accompanied by mesangial cell proliferation and mesangial matrix expansion is the most basic pathological feature of IgAN. Dihydroartemisinin (DHA), an antimalarial drug, was recently reported to be effective in treating autoimmune diseases. However, its potential therapeutic role in IgAN is relatively unstudied. The aim of this study was to investigate the pharmacological effects and the underlying mechanisms of DHA in the treatment of IgAN. In this study, renal biopsy specimens were collected for immunohistochemistry. In vitro, 25 μg/ml concentrations of aggregated IgA1 (aIgA1) was used to construct the IgAN mesangial cell model. Stimulated human mesangial cells (HMCs) were treated for 24 h with DHA (0–15 μM) and were collected for western blot analyses. Cell proliferation was assessed by Cell Counting Kit 8 (CCK8) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. In vitro, our results showed that DHA could downregulate the mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 (mTOR/S6K1) signaling pathway, promote cell autophagy, and ameliorate cell proliferation in aIgA1-induced HMCs. The results suggested that DHA may represent a novel class of mTOR inhibitor and promote an anti-proliferation effect in IgAN HMCs, which provides an alternative approach for IgAN treatment.
1. Introduction IgA nephropathy (IgAN) is the most prevalent form of glomerular kidney disease worldwide, particularly in Asian areas, and has a significant morbidity and mortality rate, with 30 to 40% of patients having impaired renal function and eventually developing end-stage renal disease (ESRD) after 20–30 years [1,2]. IgAN is characterized by immune complex deposition in the mesangial areas accompanied by mesangial cell proliferation. Mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 (mTOR/S6K1), an important cell growth coordination pathway, functions as a negative controller of autophagy which can attenuate kidney injury of IgAN [3,4]. Data revealed that the activation of the mTOR signal is a critical event to induce and accelerate the progression of IgAN [5–8]. Dihydroartemisinin (DHA) is an active metabolite compound derived from artemisinin and is widely used as an antimalarial therapeutic agent [9]. Much evidence has emerged to elucidate the biological effects of DHA, especially in immunomodulation and cell proliferation [10,11]. Based on the fact that IgAN is an autoimmune disease, glomerular mesangial cell proliferation is the most basic pathological
⁎
characteristic and is related to IgAN progression [12,13]. Thus, it is imperative to determine whether DHA has a therapeutic effect on IgAN. Here, we investigated the potential role of DHA therapy and for the first time, described the mechanism of DHA regarding the mTOR/S6K1 pathway in IgAN. Our results showed that DHA could suppress mTOR/ S6K1 signaling, induce autophagy, and inhibit proliferation of IgAN human mesangial cells (HMCs) stimulated by aggregated IgA1 (aIgA1). 2. Materials and methods 2.1. Cell culture and treatment The human glomerular mesangial cell line (CBR130735, Cellbio, China) was cultured in antibiotic-free Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Monomeric human IgA1 (Abcam) was heated and aggregated at 65℃ for 150 min on a dry plate heater to obtain aIgA1 as previously described [14]. DHA (purity > 98% by high-performance liquid chromatography) was purchased from ApexBio Technology (Houston, US), and dissolved in dimethyl sulfoxide
Corresponding author. E-mail address: [email protected] (H. Liu).
https://doi.org/10.1016/j.intimp.2019.106125 Received 30 July 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Fig. 1. Upregulation of pmTOR and pS6K1 in the kidneys of IgAN patients. (A) Representative images of glomerular immunohistostaining from IgAN patients and controls with pmTOR and pS6K1. (B) The average optical density of pmTOR and pS6K1 were analyzed by ImageJ software. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 specimens in each group).
MA), with periodic acid-Schiff (PAS) used instead of primary antibodies as negative controls for all staining. Finally, horseradish peroxidase (HRP)-conjugated polymer (Abcam, Cambridge, MA) was used for the visualized detection under light microscopy (Nikon Tokyo, Japan). OD values were analyzed by ImageJ software (Media Cybernetics, Inc., Bethesda, MD, USA). All the analyses were repeated no less than three times, and representative images are displayed. The use of renal specimens of IgAN patients was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University according to the Declaration of Helsinki. 2.4. Western blotting Total proteins were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) as previously described [16]. The lysate was centrifuged at 12,000g for 30 min at 4 °C. After centrifugation, supernatants were obtained for the evaluation of total protein concentration with bovine serum albumin (BSA) as a standard using a BCA protein assay kit (Pierce, Thermo). In brief, 30 μg of protein was used for electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% BSA at room temperature for 1 h, membranes were incubated overnight at 4 °C with the primary antibodies: pmTOR (Abcam, Cambridge, MA), pS6K1 (Abcam, Cambridge, MA), LC3B (GeneTex, Irvine, USA), and β-actin (Proteintech, Rosemont, USA). After washing with phosphate-buffered saline with Tween (PBST), blots were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG, Abcam, Cambridge, MA) at room temperature for 1 h, and enhanced chemiluminescence (Millipore) was used to visualize the bands.
Fig. 2. The construction of the IgAN HMCs model. HMCs were stimulated with different concentrations of aIgA1. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
(DMSO) to prepare a stock solution (20 mM) and stored at −20 °C. After starvation for 12 h in serum-free medium, cells were then incubated with aIgA1 alone or with a combination of DHA for 24 h. 2.2. Cell proliferation assay Cell proliferation was examined with a light microscope and accessed by Cell Counting Kit-8 (CCK8) assay (Dojindo, Kumamoto, Japan) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Servicebio, China). For CCK8, 10 μl of CCK8 was added to each well and incubated at 37 °C. Cell viability was determined by measuring optical density (OD) at a 450-nm wavelength. For MTT, 20 μl of MTT was added to each well and incubated at 37 °C. Subsequently, 200 μl of DMSO was added. The absorbance was detected at 490 nm using a spectrophotometer.
2.5. Statistics Statistical differences were analyzed by Student’s t-test, and p < 0.05 was considered statistically significant. Analyses were conducted with GraphPad Prism 6.0 statistical software.
2.3. Immunohistochemistry staining 3. Results Formaldehyde-fixed and paraffin-embedded tissue sections (4 mm thick) were used for immunohistochemistry as previously described [15]. After dehydration, slides in citrate solution were subjected to a microwave antigen retrieval process. Primary antibodies were employed against the phosphorylated mTOR (pmTOR) (Abcam, Cambridge, MA) and phosphorylated S6K1 (pS6K1) (Abcam, Cambridge,
3.1. pmTOR and pS6K1 were abnormally elevated in IgAN biopsy We had previously constructed an IgAN rat model and found that expressions of pmTOR and pS6K1 were upregulated. That is, the mTOR/S6K1 pathway was activated in IgAN. In addition, the activation 2
International Immunopharmacology 80 (2020) 106125
M. Xia, et al.
Fig. 3. DHA inhibits HMC proliferation in the IgAN HMC model. (A) The proliferation of HMCs was assessed by CCK8 assay. (B) The proliferation rate of HMCs was measured by MTT assay. (C) Treatment of aIgA1-induced HMCs without or with different concentrations of DHA for 24 h. Representative light microscopy images are presented. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
concentration-dependent manner under 10 μM, while the OD values measured by the two methods were slightly different (Fig. 3A and 3B). In addition, light microscopy of HMCs stimulated with different concentrations of DHA is shown (Fig. 3C).
of this pathway was crucial for the proliferation of mesangial cells [4]. Here, pmTOR and pS6K1 were further detected by immunohistochemistry staining in the renal biopsy section of IgAN patients. Patients with minor glomerular lesions (MCD) were used as controls. The results showed elevated expression of pmTOR and pS6K1 in renal tissue specimens of IgAN compared to MCD (Fig. 1A). Using image analysis software, the average optical density of the pmTOR and pS6K1 staining were significantly higher in IgAN patients than in controls (Fig. 1B).
3.3. DHA suppressed mTOR/S6K1 signaling Increasing evidence indicates that mTOR is a central controller of proliferation and survival, we hypothesized that DHA might disrupt cell proliferation by suppressing mTOR/S6K1 signaling. We examined the effect of DHA on the mTOR signaling pathway in IgAN HMCs in vitro. By western blot analysis, we found that DHA suppressed phosphorylation of mTOR, and consistently inhibited phosphorylation of S6K1 in a dose-response experiment, especially with 15-μM DHA treatment. In addition, we also tested autophagy representative protein LC3B expression. DHA (above 5 μM) exhibited an obvious stimulatory effect on LC3B expression (Fig. 4).
3.2. DHA diminished proliferation of aIgA1-induced mesangial cells To investigate whether DHA can inhibit the proliferation of IgAN HMCs in vitro, we used different concentrations of aIgA1 to stimulate HMCs and found the most suitable concentration (25 μg/ml) to construct an IgAN HMC model (Fig. 2). Stimulated HMCs were treated with DHA for 24 h at concentrations of 0–15 μM. The MTT and CCK8 assay results showed that DHA diminished cell proliferation in a 3
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Fig. 4. DHA reduces phosphorylation of mTOR/S6K1 signaling in IgAN. Western blot analysis for pmTOR, pS6K1, and LC3B expressions in DHA-treated IgAN HMCs. Data are presented as mean ± SD. *p < 0.05 vs control, **p < 0.01 vs control (n = 3 in each group).
4. Discussion
protein in HMCs that received DHA. This indicated that DHA had antiinflammatory and autophagy promoting effects in IgAN [33]. However, this research was only a preliminary experiment, many questions could arise from the current work. First, mTOR functions at least two complexes, mTORC1 and mTORC2. Whether DHA affects both compounds or whether it affects the complex integrity warrants further investigation and validation. Second, further research is required to determine whether DHA directly regulates the upstream molecules of mTOR, for example, phosphatidylinositol 3 kinase (PI3K). Third, more results are needed to confirm the effects of cell proliferation and autophagy, such as cell cycle experiment and electron microscope observation of autophagy flow. Reports have shown that DHA could exhibit a significant anti-proliferation effect via transferrin receptor 1 (CD71) and the mTOR/S6K1 pathway in tumors [32,34,35]. Given that CD71 also plays a key role in trapping IgA1 immune complexes in situ and mesangial proliferation in IgAN [36], we believe that DHA may decrease mesangial cell proliferation by inhibiting mTOR/S6K1 pathway and affecting IgA1 immune complex-mediated activation of CD71 expression in IgA nephropathy, which may be a new mechanism beyond affecting autophagy. However, further study is still needed to explore the potential effects of DHA on IgAN. In conclusion, the present study supports a protective role of DHA in treating IgAN by inhibiting the mTOR/S6K1 pathway, enhancing autophagy, and ameliorating mesangial cell proliferation. This provides a new clue for IgAN treatment.
In this study, the IgAN HMCs model was well-established [14], and for the first time, we described the effects of DHA on mesangial cells. We demonstrated that DHA could prevent the proliferation of mesangial cells in IgAN and through inhibition of mTOR/S6K1. Our report suggests that DHA is not only a possible mTOR inhibitor, but also has great potential for IgAN therapy. The first description of IgA nephropathy was proposed by Jean Berger in 1968, and it is also named Berger’ s disease [17]. In IgAN, increasing mesangial matrix and hypercellularity are common pathological changes, making it crucial to find ways to control mesangial cell proliferation and slow its progression. DHA is derived from artemisinin, an effective constituent originally extracted from traditional Chinese medicine Artemisia annua L [18,19]. Although artemisinin displays abundant biology effects, it has attracted increasing attention in treating lupus in recent decades [20–24]. However, only studies have examined DHA in renal disease [25–28]. Thus, the possible role of DHA in nephropathy remains unclear. Increasing evidence demonstrates that DHA can potently inhibit cell proliferation, in particular, through the proteins regulated by mTOR, such as FAK, MMP-2, and NF-κB [29–31]. In addition, research has also shown that DHA could potently inhibit mTORC1-mediated pS6K1 in the tumor cells [32]. These led us to investigate whether DHA primarily inhibits mTOR signaling and then affects its downstream. In the previous study, we found that mTOR-mediated autophagy inhibition may result in mesangial cell proliferation in IgAN, while the inhibitor of mTOR could enhance repressed autophagy and attenuates aggressive progression in a rat model of IgAN [4]. Here, we hypothesized that DHA is able to affect IgAN through a similar mechanism. Our results showed a significant anti-proliferation effect on HMCs when treated with DHA (5–15 μM). In addition, our results also showed downregulation in phosphorylation of mTOR/S6K1 and upregulation of LC3B autophagy
CRediT authorship contribution statement Ming Xia: Investigation, Methodology, Writing - original draft. Di Liu: Software, Validation. Xiaofang Tang: Software, Formal analysis. Yexin Liu: Writing - review & editing. Haiyang Liu: Data curation. Yu Liu: Project administration, Funding acquisition. Guochun Chen: 4
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Conceptualization, Writing - review & editing. Hong Liu: Supervision, Funding acquisition.
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Acknowledgments We sincerely appreciate the time and effort of all who contributed to this study. Funding This work was supported by research grants (81770714), (81470947), and (81570622) from the National Natural Science Foundation of China. It was also funded by the postgraduate innovation project of Central South University in China (Project no. 2018zzts920). Declaration of Competing Interest No conflicts of interest, financial or otherwise, are declared by the authors. References [1] K.N. Lai, et al., IgA nephropathy, Nat. Rev. Dis. Primers 2 (2016) 16001. [2] S.J. Barbour, et al., Individuals of Pacific Asian origin with IgA nephropathy have an increased risk of progression to end-stage renal disease, Kidney Int. 84 (5) (2013) 1017–1024. [3] R.A. Saxton, D.M. Sabatini, mTOR signaling in growth, metabolism, and disease, Cell 168 (6) (2017) 960–976. [4] D. Liu, et al., Rapamycin enhances repressed autophagy and attenuates aggressive progression in a rat model of IgA nephropathy, Am. J. Nephrol. 45 (4) (2017) 293–300. [5] R.C. Monteiro, et al., Pathogenic significance of IgA receptor interactions in IgA nephropathy, Trends Mol. Med. 8 (10) (2002) 464–468. [6] H. Tamouza, et al., Transferrin receptor engagement by polymeric IgA1 induces receptor expression and mesangial cell proliferation: role in IgA nephropathy, Contrib. Nephrol. 157 (2007) 144–147. [7] J. Tian, et al., The Akt/mTOR/p70S6K pathway is activated in IgA nephropathy and rapamycin may represent a viable treatment option, Exp. Mol. Pathol. 99 (3) (2015) 435–440. [8] H. Tamouza, et al., The IgA1 immune complex-mediated activation of the MAPK/ ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy, Kidney Int. 82 (12) (2012) 1284–1296. [9] Y. Li, Qinghaosu (artemisinin): chemistry and pharmacology, Acta Pharmacol. Sin. 33 (9) (2012) 1141–1146. [10] L. Bai, et al., Immunosuppressive effect of artemisinin and hydroxychloroquine combination therapy on IgA nephropathy via regulating the differentiation of CD4+ T cell subsets in rats, Int. Immunopharmacol. 70 (2019) 313–323. [11] R. Lin, et al., Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells, Cancer Lett. 381 (1) (2016) 165–175. [12] J. Novak, et al., Glycosylation of IgA1 and pathogenesis of IgA nephropathy, Semin. Immunopathol. 34 (3) (2012) 365–382.
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