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Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy
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Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy Dianwen Xu1, Juxiong Liu1, He Ma, Wenjin Guo, Jiaxin Wang, Xingchi Kan, Yanwei Li, ⁎ Qian Gong, Yu Cao, Ji Cheng, Shoupeng Fu College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Schisandrin A Mastitis Nrf2 Autophagy mTOR AMPK-ULK1
Schisandrin A (Sch A), a dibenzocyclooctadiene lignan extracted from Schisandra chinensis (Turcz.) Baill., has anti-oxidant and anti-inflammatory effects, but the effect on masitits has not been studied. Therefore, we investigated the effect of Sch A in cell and mouse models of lipopolysaccharide (LPS)-induced mastitis. Studies in vivo showed that Sch A reduced LPS-induced mammary injury and the production of pro-inflammatory mediators. Sch A also decreased the levels of pro-inflammatory mediators and activated nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway in mouse mammary epithelial cells (mMECs). The Nrf2 inhibitor partially abrogated the downregulation of Sch A on LPS-induced inflammatory response. In addition, LPS stimulation suppressed autophagy, while both Sch A and the autophagy inducer rapamycin activated autophagy in mMECs, which down-regulated inflammatory response. Sch A also restrained LPS-induced phosphorylation of mammalian target of rapamycin (mTOR) and activated AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1). In summary, these results suggest that Sch A exerts protective effects in LPS-induced mastitis models by activating Nrf2 signaling pathway and inducing autophagy and the autophagy is initiated by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway.
1. Introduction Mastitis refers to the inflammation of mammary gland that not only affects the health benefits of breastfeeding in lactating women but is also one of the most frequent and costly diseases in the dairy industry [1–4]. Mastitis is mainly caused by bacterial infections, including Gram-negative bacteria [5], such as Escherichia coli, which has been documented as one of the major etiological agents of mastitis [6]. It is typically characterized by an infiltration of somatic cells, mainly polymorphonuclear cells, into mammary tissues [7]. Although the pathogenesis of mastitis has not been fully elucidated, studies have shown that oxidative stress and autophagy may be involved [7,8]. The inflammatory response is associated with oxidative stress [9]. Mastitis is accompanied by a systemic oxidative stress response [10]. Oxidative stress process can kill pathogenic microorganisms, but it can also destroy various biomolecules and cause damage to mammary tissues [11]. Therefore, the body has developed various antioxidant mechanisms to counterbalance oxidative stress, such as nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway and macroautophagy
(hereafter referred to as autophagy) [12–15]. The Nrf2 pathway is a major regulator of cellular oxidative stress and protects cells from oxidative stress damage [16]. Under normal physiological conditions, the transcription factor Nrf2 interacts with its inhibitory protein Kelchlike ECH-associated protein 1 (Keap-1) and located in the cytoplasm. Upon exposure to stressors, Nrf2 is disconnected from Keap-1 and translocates into the nucleus to transactivate the transcription of antioxidant genes and cytoprotective genes, such as autophagy-related genes, thereby promoting cell survival [17–19]. However, in Escherichia coli-induced acute mastitis, the body's oxidative stress levels may overwhelm the antioxidant defense mechanisms, thereby aggravating inflammation and causing extensive mammary damage [11]. In addition, studies have found that autophagy, as a lysosomal-dependent protein degradation pathway [20], can inhibit inflammation by limiting activation of the inflammasomes and suppressing the secretion of proinflammatory cytokines [21,22]. Moreover, impaired autophagy is involved in the susceptibility of cows to mastitis [8], suggesting that autophagy may have an important effect on mastitis. Therefore, therapeutic strategies for regulating the body's antioxidant capacity and
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Corresponding author. E-mail address: [email protected] (S. Fu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.intimp.2019.105983 Received 16 August 2019; Received in revised form 11 October 2019; Accepted 14 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Dianwen Xu, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105983
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previously described [32]. Briefly, mammary tissues were homogenized with an appropriate amount of HEPES (N-2-hydroxyethylpiperazine-Nethane-sulphonicacid) solution and the supernatant was isolated for ELISA after ultracentrifugation. Subsequently, the precipitate was homogenized again with 0.5% CTAC (Cetyltrimethylammonium chloride) equivalent to HEPES and the supernatant was collected for MPO activity assay after centrifugation. 75 μL of sample was added into a 96-well plate after 10-fold dilution with 5% CTAC and each sample had three replicates. After that, 75 μL of substrate was added into the plate, and 2–3 min later, the reaction was terminated with 100 μL of 2 mol/L sulfuric acid. Finally, the absorbance at 450 nm was determined.
autophagy progression may be effective in treating mastitis. Schisandrin A (Sch A) is a biologically active dibenzocyclooctadiene lignan extracted from traditional Chinese herbal medicine Schisandra chinensis (Turcz.) Baill. [23]. A growing number of studies indicate that Sch A has anti-oxidant and anti-inflammatory effects [24–27]. However, whether Sch A can protect against lipopolysaccharide (LPS)-induced mastitis is unknown. Therefore, our study investigated the effect of Sch A in cell and mouse models of LPS-induced mastitis and its underlying mechanisms. 2. Materials and methods 2.1. Reagents
2.5. Determination of cytokine levels
Schisandrin A (Sch A, purity ≥ 98%) was bought from Shanghai YuanYe Biotechnology (Shanghai, China). Lipopolysaccharides from Escherichia coli O55:B5, all-trans-retinoic acid (ATRA) (purity ≥ 98%) and Compound C (purity ≥ 98%) were bought from Sigma-Aldrich (St Louis, MO, U SA). Rapamycin (Rap, purity = 99.30%) was obtained from Selleckchem (Houston, DE, USA). The primary antibodies against p-AMPKα (Thr172) (#2535), p-ERK1/2 (Thr202/Tyr204) (#9101), pAKT (Ser473) (#4070), p-mTOR (Ser2448) (#5536) and Histone H3 (#4499), AMPKα (#5832), mTOR (#2972), p-NF-κB p65 (#3033S), NF-κB p65 (#8242) and p-ULK1 (#5869T) were provided by Cell Signaling Technology (Beverly, MA, USA). Antibodies against ERK1/2 (16443-1-AP), AKT (10176-2-AP), ULK1 (20986-I-AP), LC3B (18725-IAP) and p62/SQSTM1 (18420-I-AP) were purchased from Proteintech (Wuhan, China). Antibodies against Nrf2 (NFE2L2) (ab31163), HO-1 (Heme Oxygenase-1) (ab68477), COX-2 (cyclooxygenase-2) (ab62331) and iNOS (inducible nitric oxide synthase) (ab15323) were purchased from Abcam (Cambridge, UK). β-Tubulin was bought from Beijing Ray Antibody Biotechnology (Beijing, China).
The content of cytokines TNF-α and IL-1β in tissues was measured by the enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Biolegend, San Diego, CA, USA) following the manufacturer’s instructions. 2.6. Cell culture Mouse mammary epithelial cells (mMECs) were obtained from the American Type Culture Collection (ATCC, ATCC®CRL-3063™) and maintained in DMEM medium (Gibco, Grand Island, NY 14072, USA) containing 10% fetal bovine serum (Clark Bioscience, Richmond, VA, USA) in a 37 °C incubator with 5% carbon dioxide. 2.7. CCK-8 assay The Cell Counting Kit-8 (CCK-8) (Saint-Bio, Shanghai, China) was applied to test whether Sch A affected cell viability. The mMECs were cultured in a 96-well plate for 12 h. After cultured in serum-free medium for 3 h, the cells were administrated with different molar concentrations of Sch A (40, 60, 80 and 100 μM) for 1 h, followed by exposure to LPS for 4 h. Subsequently, the cells in each well were incubated for 1 h after adding 10 μL of CCK-8 solution. Finally, the absorbance at 450 nm was determined.
2.2. Animal and treatment C57BL/6 mice (6–8 weeks old, 25–30 g) were provided by the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were kept in stainless, certified cages with good care conditions during experiments. In this study, all experiments were ratified by the Jilin University Institutional Animal Care and Use Committee and followed the guidelines (Protocol No. 2015047) established on 27 February 2015. One week after delivery, 30 female mice were stochastically assigned to 5 groups: Control group, Sch A group, LPS treatment group, LPS + Sch A group, and LPS + Dexamethasone (Dex) group with 6 mice in each group. Mice were administrated by intraperitoneal injection with 32 mg/kg of Sch A [28,29] or 5 mg/kg of Dex [30], respectively. One hour later, LPS was infused into the fourth pair of mammary glands via ducts. After 12 h, mice were administrated with 32 mg/kg of Sch A or 5 mg/kg of Dex, respectively, for another 12 h. Finally, the mice euthanized by cervical dislocation and the mammary glands were separated and stored as follow-up experimental samples.
2.8. Quantitative real-time PCR (qRT-PCR) Total RNA was separated from mMECs by using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and then was reverse-transcripted into cDNA by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Quantitative RT-PCR was performed with the SYBR Green QuantiTect RT-PCR Kit (Roche, South San Francisco, CA, USA). The primers were designed by Sangon Biotech (Shanghai, China) and the sequences are shown in Table 1. In this experiment, β-actin was selected as an internal reference gene and the relative mRNA levels of target genes were obtained by the 2−ΔΔCt method.
2.3. Assessment of histopathological changes
Table 1 The primer sequences of IL-1β, TNF-α, HO-1 and β-actin.
The mammary tissue blocks were fixed in 4% formaldehyde solution for 24 h, then embedded in paraffin, and finally made into 5 μm sections. Subsequently, to evaluate pathological changes, the sections were objected to staining with hematoxylin-eosin (H&E), observed and photographed under a light microscope. Finally, the pathological changes were determined with reference to the scores for injury degree as previously described [31]. 2.4. Determination of myeloperoxidase (MPO) activity The levels of MPO were determined according to the method 2
Gene
Sequences
Length (bp)
IL-1β
(F) 5′-GCAGAGCACAAGCCTGTCTTCC-3′ (R) 5′-ACCTGTCTTGGCCGAGGACTAAG-3′
198
TNF-α
(F) 5′-GCGACGTGGAACTGGCAGAAG-3′ (R) 5′-GCCACAAGCAGGAATGAGAAGAGG-3′
103
HO-1
(F) 5′-ACCGCCTTCCTGCTCAACATTG-3′ (R) 5′-CTCTGACGAAGTGACGCCATCTG-3′
104
β-actin
(F) 5′-ATCACTATTGGCAACGAGCGGTTC-3′ (R) 5′- CAGCACTGTGTTGGCATAGAGGTC-3′
156
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Fig. 1. Schisandrin A (Sch A) alleviates mammary injury in lipopolysaccharide (LPS)-induced mouse mastitis. Mice were pretreated with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treatment with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for H&E staining. (A) Control; (B) Sch A (32 mg/kg); (C) LPS (0.2 mg/kg); (D) LPS + Sch A (32 mg/kg); (E) LPS + Dex (5 mg/kg); (F) Mammary gland injury scores. Values are presented as mean ± SD (n = 6). The black arrows indicate the inflammatory cell infiltration in mammary tissues. Dex served as a positive control. Scale bar: 50 μm. ####P < 0.0001 vs. Control group and ****P < 0.0001 vs. LPS-treated group.
membrane (Millipore, Darmstadt, Germany) at 75 V for 1 h. The transferred membranes were blocked in TBS-T containing 5% skim milk for 2 h, followed by incubation overnight at 4 °C with each primary antibody in TBS-T containing 5% bovine serum albumin (Gentihold, Beijing, China). After washing five times with TBS-T, the PVDF membranes were co-incubated with the corresponding HRP-labeled secondary antibodies (1:3000 dilution) (Bosterbio, USA) at room temperature for 1 h. Finally, the immunoreactive blots were visualized with an enhanced chemiluminescent substrate (Beyotime, Shanghai, China). 2.10. Statistical analysis In this study, all data analysis work was performed using GraphPad Prism 6.0 software (La Jolla, CA, USA). The final results were presented in the form of mean ± SD. Statistical differences between groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons (P values < 0.05).
Fig. 2. Sch A down-regulates myeloperoxidase (MPO) activity in vivo. Mice were pretreated with Sch A (32 mg/kg) or Dex (5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for MPO activity assay. Values are presented as mean ± SD (n = 6). Dex served as a positive control. ####P < 0.0001 vs. Control group and ****P < 0.0001 vs. LPS-treated group.
3. Results 3.1. Sch A alleviates mammary injury in vivo
2.9. Western blotting To evaluate the effect of Sch A on mastitis, we constructed an LPSinduced mouse mastitis model. Mice were pretreated with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treating with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for H&E staining. The results showed that in comparison with the control group, the number of inflammatory cells infiltrating into the mammary gland cavity was remarkably increased by LPS treatment (Fig. 1A-C). However, treatment with Sch A (32 mg/ kg) or Dexamethasone (Dex, 5 mg/kg) significantly lowered LPS-induced infiltration of inflammatory cells (Fig. 1D-F). These results indicate that Sch A alleviates mammary injury in LPS-induced mouse
To obtain the total protein, homogenized mouse mammary tissues and treated mMECs were lysed by the addition of lysis buffer (Beyotime, Shanghai, China), followed by centrifugation for 15 min at 12,000g. The supernatants containing the total protein were collected. Similarly, cytoplasmic and nuclear proteins were obtained by applying the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Subsequently, the protein concentrations were determined by using the BCA Protein Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Protein samples were loaded onto 6%-15% SDS-PAGE at 110 V for 90 min and then transferred onto a PVDF 3
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Fig. 3. Sch A reduces the production of pro-inflammatory mediators in vivo. Mice were pretreated with Sch A (32 mg/kg) or Dex (5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treatment with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h. Dex served as a positive control. (A-B) The protein levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). (C) The protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were determined by western blotting. (D-E) Bar graphs represent quantitative results for corresponding protein bands. Values are presented as mean ± SD (n = 6). ####P < 0.0001 vs. Control group; ****P < 0.0001 vs. LPS-treated group.
3.4. Sch A inhibits LPS-induced inflammatory response in mMECs
mastitis.
Before evaluating the role of Sch A, the cytotoxicity was determined by CCK-8 assay. The result showed that Sch A at concentrations of 40, 60, 80 and 100 μM did not alter the cell viability with or without LPS stimulation (5 μg/mL) (Fig. 4A). Subsequently, the anti-inflammatory effect of Sch A was evaluated. The qRT-PCR analysis showed that stimulation with LPS in mMECs induced over-expression of IL-1β and TNFα mRNA, which was reduced by Sch A at 10, 20 and 40 μM (Fig. 4B and C). Similarly, LPS treatment remarkably elevated the expressions of COX-2 and iNOS, which also was reduced by Sch A at 10, 20 and 40 μM (Fig. 4D-F). These results demonstrate that Sch A mitigates inflammatory response in LPS-stimulated mMECs.
3.2. Sch A down-regulates MPO activity in vivo At the same time, considering that myeloperoxidase (MPO) is an important indicator of neutrophil infiltration into tissues, oxidative stress and tissue damage [33], the levels of MPO in mammary tissues were determined. The results showed that in comparison with the control group, MPO activity was remarkably elevated by LPS treatment. However, treatment with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) remarkably reduced MPO activity (Fig. 2). These results indicate that Sch A treatment significantly inhibits LPS-induced increase in MPO activity.
3.3. Sch A mitigates inflammatory response in vivo
3.5. The Nrf2 signaling pathway is involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs
The ELISA analysis showed that stimulation with LPS in mMECs increased the levels of IL-1β and TNF-α, which were reduced by Sch A treatment at 32 mg/kg (Fig. 3A and B). Similarly, LPS treatment remarkably elevated the protein expressions of COX-2 and iNOS, which were reduced by Sch A treatment (32 mg/kg) (Fig. 3C-E). Taken together, our data demonstrate that Sch A mitigates LPS-induced inflammatory response in vivo.
To evaluate the possibility that Nrf2 pathway was involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs, the mMECs were treated with Sch A (40 μM) at different time points (0, 1, 3, 6 and 12 h). Subsequently, the cells were collected for western blotting. The results showed that the levels of nuclear Nrf2 and Heme Oxygenase-1 (HO-1) were significantly increased with time (Fig. 5A-C). Although Sch A was proved to activate Nrf2 pathway, it was still 4
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Fig. 4. Sch A mitigates LPS-induced inflammatory response in vitro. (A) After serum starvation for 3 h, mouse mammary epithelial cells (mMECs) were administrated with different molar concentrations of Sch A (40, 60, 80 and 100 μM) for 1 h, followed by treatment with LPS for 4 h. Then, cell viability was determined by Cell Counting Kit-8 (CCK-8). (B-C) After serum starvation for 3 h, mMECs were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h, and then administrated with LPS for 4 h. Subsequently, the mRNA levels of IL-1β and TNF-α were detected by quantitative real-time PCR (qRT-PCR). (D) The protein levels of COX-2 and iNOS were determined by western blotting. (E-F) Bar graphs represent quantitative results for corresponding protein bands. Values are expressed as mean ± SD (n = 3). ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. LPS-treated group.
in LPS-stimulated mMECs. Although Sch A was proved to enhance autophagy, it was still unknown if autophagy was involved in the antiinflammatory effect of Sch A in LPS-stimulated mMECs. To evaluate the effect of autophagy, mMECs were treated with the autophagy inducer rapamycin (a specific mTOR inhibitor) for 2 h before LPS stimulation. After that, we observed that treatment with rapamycin increased the amount of LC3 II protein, lowered the amount of p62 protein (Fig. 6DF) and down-regulated the levels of IL-1β and TNF-α mRNA (Fig. 6I-J). In addition, both Sch A and rapamycin reduced phosphorylation of NFκB p65 (Fig. 6G-H). These results indicate that Sch A mitigates LPSinduced inflammatory response in mMECs, at least partially, due to autophagy induction.
unknown if Nrf2 pathway was involved in the roles of Sch A in LPSstimulated mMECs. Therefore, mMECs were treated with an Nrf2 inhibitor all-trans-retinoic acid (ATRA, 2 μM) for 6 h and then administrated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/ ml) for 4 h. After that, under the condition that Nrf2 was inhibited (Fig. 5D), Sch A still reduced the expression of TNF-α mRNA (Fig. 5F), but lost the inhibitory effect on the expression of IL-1β mRNA (Fig. 5E). These results indicate that Nrf2 inhibition partially abrogates the antiinflammatory effect of Sch A in LPS-stimulated mMECs, which means that Nrf2 pathway is partially responsible for the protective effect of Sch A in LPS-stimulated mMECs. 3.6. Sch A prevents LPS-induced inflammatory response by inducing autophagy in LPS-stimulated mMECs
3.7. Sch A induces autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs
To study if autophagy occurs in LPS-stimulated mMECs in the presence of Sch A, the autophagy makers LC3B (microtubule-associated protein 1 light chain 3 β) and p62 (SQSTM1/sequestosome 1) were determined by western blotting. Compared with the control group, LC3 II protein was decreased and p62 protein was increased after stimulation with LPS. However, treatment with Sch A at 10, 20 and 40 μM all elevated the amount of LC3 II protein and reduced the amount of p62 protein (Fig. 6A-C). The results indicate that Sch A induces autophagy
The mammalian target of rapamycin (mTOR) is a key negative regulator of autophagy activity [34]. To elucidate the molecular mechanism by which Sch A induced autophagy, we first evaluated the effect of Sch A on the phosphorylation status of mTOR. The results showed that LPS treatment elevated the level of p-mTOR, which was reduced by Sch A at 10, 20 and 40 μM (Fig. 7A and E). Subsequently, we determined the phosphorylation status of the upstream kinases protein 5
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Fig. 5. The nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway is involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs. (A) mMECs were treated with 40 μM Sch A at different time points (0, 1, 3, 6 and 12 h). The protein levels of Nuclear-Nrf2 and Heme Oxygenase-1 (HO-1) were detected by western blotting. (B-C) Bar graphs represent quantitative results for corresponding protein bands. (D-E) mMECs were pretreated with a Nrf2 inhibitor all-transretinoic acid (ATRA, 2 μM) for 6 h and then treated with Sch A (40 μM) for 1 h , followed by exposure to LPS (5 μg/mL) for 4 h. The mRNA levels of IL-1β and TNF-α were determined by qRT-PCR. Values are presented as mean ± SD (n = 3). ##P < 0.01, ###P < 0.001 and ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. LPS-treated group; $P < 0.05 and $$$$P < 0.0001 vs. Sch A (40 μM) and LPS (5 μg/mL) co-treated group.
evaluated the effect of Sch A on the level of p-ULK1 (Ser555). The results showed that LPS treatment reduced the level of p-ULK1 (Ser555), which was elevated by Sch A at 10, 20 and 40 μM (Fig. 7F and G). To confirm that AMPK activation is responsible for the effect of Sch A on the level of p-ULK1 (Ser555), cells were pretreated with Compound C (CC, a potent inhibitor of AMPK, 10 μM) for 2 h and then administrated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/ml) for 4 h. The results showed that Sch A-induced phosphorylation of ULK1 at Ser555 was blocked by AMPK inhibitor (Fig. 7H-J). Collectively, these
kinase B (AKT), AMP-activated protein kinase (AMPK) and extracellular regulated protein kinase (ERK1/2). Among them, AMPK activation, AKT inhibition and ERK1/2 inhibition can all lead to inactivation of mTOR [35]. Our data showed that only the trends of p-AKT and pAMPK were consistent with that of p-mTOR (Fig. 7A, B, D and E), although the phosphorylation levels of the three kinases changed after treatment with Sch A (Fig. 7A-D). In addition, considering that AMPK can directly activate autophagy by phosphorylating unc-51 like kinase 1 (ULK1) at Ser 555 [36,37], we
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Fig. 6. Sch A prevents LPS-induced inflammatory response by inducing autophagy in LPS-stimulated mMECs. After serum starvation for 3 h, cells were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h or Rap (a specific inhibitor of mTOR, 200 nM) for 2 h, followed by treatment with LPS (5 μg/ mL) for 4 h. (A and D) The protein levels of LC3B and p62 were detected by western blotting. (B-C) and (E-F) Bar graphs represent quantitative results for corresponding protein bands. (G) The protein levels of p65 and p-p65 were detected by western blotting. (H) Bar graphs represent quantitative results for corresponding protein bands. (I-J) The mRNA levels of IL-1β and TNF-α were detected by qRT-PCR. Values are presented as mean ± SD (n = 3). ##P < 0.01, ### P < 0.001 and ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. LPS-treated group.
caused by pathogenic bacteria [1]. The typical pathological characteristic of mastitis is a large number of somatic cells, mainly neutrophils infiltrating into the mammary tissues [7]. In this study, we observed that Sch A treatment remarkably reduced LPS-induced infiltration of inflammatory cells into mammary tissues. Consistent with this notion, Sch A remarkably inhibited LPS-induced increase in MPO activity, a representative indicator of neutrophils infiltration into tissues, oxidative stress and tissue damage [33]. In addition, when exposed to inflammatory stimuli, such as LPS, cells produce excessive pro-inflammatory mediators such as IL-1β, TNF-α, COX-2 and iNOS, resulting in damage to mammary tissues [7]. In our study, both in vivo and in vitro experiments indicate that Sch A can mitigate LPS-induced mastitis. As mentioned earlier, because MPO activity can reflect the degree of oxidative stress, the results of MPO activity analysis indicate that Sch A may have a regulatory effect on oxidative stress in LPS-induced
results suggest that Sch A induces autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs. 4. Discussion Several pharmacological activities of Sch A have been demonstrated in previous studies, but whether Sch A has protective effects on mastitis is unknown. In this study, we showed that Sch A inhibits LPS-induced inflammatory response via activating Nrf2 signaling pathway and inducing autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway, which reduced mammary tissue damage and the production of pro-inflammatory mediators, ultimately inhibiting LPS-induced mouse mastitis. Mastitis refers to the inflammation of mammary gland mainly 7
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Fig. 7. Sch A activates autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs. After serum starvation for 3 h, cells were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h, followed by treatment with LPS for 4 h. (A) The protein levels of p-mTOR, mTOR, p-AMPK, AMPK, p-ERK1/2, ERK1/2, p-AKT and AKT were determined by western blotting. (B-E) Bar graphs represent quantitative results for corresponding protein bands. (F) The protein levels of p-ULK1 and ULK1 were determined by western blotting. (G) Bar graphs represent quantitative results for corresponding protein bands. (H) mMECs were pretreated with Compound C (CC, 10 μM) for 2 h and then treated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/mL) for 4 h. The protein levels of p-AMPK, AMPK, p-ULK1 and ULK1 were determined by western blotting. (I-J) Bar graphs represent quantitative results for corresponding protein bands. Values are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. LPS-treated group; $$$$P < 0.0001 vs. Sch A (40 μM) and LPS (5 μg/mL) co-treated group.
These results indicate that Nrf2 pathway partially contributes to the protective effect of Sch A in LPS-stimulated mMECs. However, because Nrf2 was not required for the inhibitory effect of Sch A on the expression of IL-1β mRNA, the underlying protective mechanisms of Sch A need to be further investigated. Autophagy is a process that relies on lysosomal degradation of proteins and organelles and is critical for cell survival and homeostasis maintenance [13]. Studies show that autophagy exerts the protective effects in several inflammatory diseases [38–41]. It is reported that impaired autophagy may be responsible for the increased susceptibility of cows to mastitis [8] and Sch A can induce autophagy in an acute lung injury model [26], which prompted us to investigate the possibility that Sch A suppresses LPS-induced mastitis by inducing autophagy. In this
mastitis. In response to sub-lethal stress such as oxidative stress, cells quickly adapt to adverse conditions through various stress response pathways to protect themselves from damage [12]. Among them, the transcription factor Nrf2 (nuclear factor-E2 associated factor 2) can transactivate the expression of antioxidant enzymes, such as Heme Oxygenase-1 (HO-1) [17]. These antioxidant enzymes can scavenge excess reactive oxygen species, thereby inhibiting the inflammatory response [38]. Sch A can activate Nrf2 signaling pathway in LPS-stimulated RAW 264.7 macrophages [27], consistently, our data suggest that Sch A can activate Nrf2 signaling pathway and induce HO-1 expression in mMECs. In addition, we observed that under the condition that Nrf2 was inhibited, Sch A still reduced the expression of TNF-α mRNA, but lost the inhibitory effect on the expression of IL-1β mRNA.
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study, for the first time, we showed that LPS stimulation suppresses autophagy [40,41] and Sch A treatment induces a protective autophagic process to inhibit LPS-induced mouse mammary epithelial inflammation. NF-κB is an important nuclear transcription factor that can transactivate the transcription of many pro-inflammatory genes [42]. LPS stimulation can induce NF-κB activation [43] and autophagy induction can suppress NF-κB activation by degrading p-NF-κB [44]. We investigated the relationship between autophagy induction and NF-κB down-regulation. In our study, when autophagy was induced, the level of p-NF-κB p65 was significantly reduced. This study provides a perspective to explain the possible mechanism of the protective effect of Sch A in LPS-induced mastitis, in which autophagy may play an essential role. Furthermore, we further investigated the possible mechanism of autophagy induction by Sch A. The kinase mTOR is the pivotal negative regulator of autophagy, and its activity is regulated by some upstream signaling pathways [38]. Previous studies have shown that the upstream signaling pathways involved in the regulation of mTOR activity mainly include AKT, AMPK and ERK1/2, among which AKT inhibition, ERK1/2 inhibition, and AMPK activation can all initiate autophagy [35]. Our results showed that only the trends of p-AKT and p-AMPK were consistent with that of p-mTOR, although the phosphorylation levels of the three kinases changed after treatment with Sch A. In addition, studies found that AMPK can directly activate autophagy by phosphorylating ULK1 at multiple sites, including Ser555 [36,37]. Consistent with this finding, AMPK activation is responsible for Sch Ainduced phosphorylation of ULK1 at Ser555. These results indicate that Sch A treatment inactivates mTOR signaling pathway and activates AMPK-ULK1 signaling pathway. These, in turn, promote autophagy initiation and mitigate LPS-induced inflammatory response. In summary, our results demonstrate that Sch A can attenuate mammary tissue damage and lower the production of pro-inflammatory mediators. These protective effects are mediated by activation of Nrf2 pathway and induction of autophagy. Moreover, the autophagy is initiated at least partially by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway. Taken together, our findings suggest that Sch A exerts a protective effect in cell and mouse models of LPS-induced mastitis and is promising for use in the treatment of mastitis.
References [1] A. Bradley, Bovine mastitis: an evolving disease, Vet. J. 164 (2) (2002) 116–128. [2] Y. Zhu, G. Liu, X. Du, Z. Shi, M. Jin, X. Sha, X. Li, Z. Wang, X. Li, Expression patterns of hepatic genes involved in lipid metabolism in cows with subclinical or clinical ketosis, J. Dairy Sci. (2018). [3] W. Yang, H. Zerbe, W. Petzl, R.M. Brunner, J. Gunther, C. Draing, S. von Aulock, H.J. Schuberth, H.M. Seyfert, Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NFkappaB in mammary epithelial cells and to quickly induce TNFalpha and interleukin-8 (CXCL8) expression in the udder, Mol. Immunol. 45 (5) (2008) 1385–1397. [4] M. Marin, R. Arroyo, I. Espinosa-Martos, L. Fernandez, J.M. Rodriguez, Identification of Emerging Human Mastitis Pathogens by MALDI-TOF and Assessment of Their Antibiotic Resistance Patterns, Front. Microbiol. 8 (2017) 1258. [5] J.L. Watts, Etiological agents of bovine mastitis, Vet. Microbiol. 16 (1) (1988) 41–66. [6] S. Passey, A. Bradley, H. Mellor, Escherichia coli isolated from bovine mastitis invade mammary cells by a modified endocytic pathway, Vet. Microbiol. 130 (1–2) (2008) 151–164. [7] X. Zhao, P. Lacasse, Mammary tissue damage during bovine mastitis: causes and control, J. Anim. Sci. 86 (13 Suppl) (2008) 57–65. [8] M. Sugimoto, Y. Sugimoto, Variant in the 5' untranslated region of insulin-like growth factor 1 receptor is associated with susceptibility to mastitis in cattle, G3 (Bethesda) 2 (9) (2012) 1077–1084. [9] Q. Chi, X. Chi, X. Hu, S. Wang, H. Zhang, S. Li, The effects of atmospheric hydrogen sulfide on peripheral blood lymphocytes of chickens: Perspectives on inflammation, oxidative stress and energy metabolism, Environ. Res. 167 (2018) 1–6. [10] R. Turk, C. Piras, M. Kovacic, M. Samardzija, H. Ahmed, M. De Canio, A. Urbani, Z.F. Mestric, A. Soggiu, L. Bonizzi, P. Roncada, Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis, J. Proteomics 75 (14) (2012) 4412–4428. [11] K. Lauzon, X. Zhao, A. Bouetard, L. Delbecchi, B. Paquette, P. Lacasse, Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage, J. Dairy Sci. 88 (12) (2005) 4295–4303. [12] G. Kroemer, G. Marino, B. Levine, Autophagy and the integrated stress response, Mol. Cell 40 (2) (2010) 280–293. [13] K. Taguchi, N. Fujikawa, M. Komatsu, T. Ishii, M. Unno, T. Akaike, H. Motohashi, M. Yamamoto, Keap1 degradation by autophagy for the maintenance of redox homeostasis, Proc. Natl. Acad. Sci. U S A 109 (34) (2012) 13561–13566. [14] R. Sumpter Jr., B. Levine, Autophagy and innate immunity: triggering, targeting and tuning, Semin. Cell Dev. Biol. 21 (7) (2010) 699–711. [15] M. Battino, F. Giampieri, F. Pistollato, A. Sureda, M.R. de Oliveira, V. Pittala, F. Fallarino, S.F. Nabavi, A.G. Atanasov, S.M. Nabavi, Nrf2 as regulator of innate immunity: A molecular Swiss army knife!, Biotechnol. Adv. 36 (2) (2018) 358–370. [16] K. Takaya, T. Suzuki, H. Motohashi, K. Onodera, S. Satomi, T.W. Kensler, M. Yamamoto, Validation of the multiple sensor mechanism of the Keap1-Nrf2 system, Free Radical. Bio Med. 53 (4) (2012) 817–827. [17] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [18] M. Pajares, N. Jimenez-Moreno, A.J. Garcia-Yague, M. Escoll, M.L. de Ceballos, F. Van Leuven, A. Rabano, M. Yamamoto, A.I. Rojo, A. Cuadrado, Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes, Autophagy 12 (10) (2016) 1902–1916. [19] S. Dayalan Naidu, D. Dikovskaya, E. Gaurilcikaite, E.V. Knatko, Z.R. Healy, H. Mohan, G. Koh, A. Laurell, G. Ball, D. Olagnier, L. de la Vega, I.G. Ganley, P. Talalay, A.T. Dinkova-Kostova, Transcription factors NRF2 and HSF1 have opposing functions in autophagy, Sci. Rep. 7 (1) (2017) 11023. [20] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (1) (2008) 27–42. [21] M. Takahama, S. Akira, T. Saitoh, Autophagy limits activation of the inflammasomes, Immunol. Rev. 281 (1) (2018) 62–73. [22] T. Monkkonen, J. Debnath, Inflammatory signaling cascades and autophagy in cancer, Autophagy 14 (2) (2018) 190–198. [23] A. Szopa, R. Ekiert, H. Ekiert, Current knowledge of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) as a medicinal plant species: a review on the bioactive components, pharmacological properties, analytical and biotechnological studies, Phytochem. Rev. 16 (2) (2017) 195–218. [24] F. Song, K. Zeng, L. Liao, Q. Yu, P. Tu, X. Wang, Schizandrin A Inhibits microgliamediated neuroninflammation through inhibiting TRAF6-NF-kappaB and Jak2Stat3 signaling pathways, PLoS One 11 (2) (2016) e0149991. [25] Y.H. Choi, Schizandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells, Biomed. Pharmacother. 106 (2018) 902–909. [26] Y. Lu, W.J. Wang, Y.Z. Song, Z.Q. Liang, The protective mechanism of schisandrin A in d-galactosamine-induced acute liver injury through activation of autophagy, Pharm. Biol. 52 (10) (2014) 1302–1307. [27] D.H. Kwon, H.J. Cha, E.O. Choi, S.H. Leem, G.Y. Kim, S.K. Moon, Y.C. Chang, S.J. Yun, H.J. Hwang, B.W. Kim, W.J. Kim, Y.H. Choi, Schisandrin A suppresses lipopolysaccharide-induced inflammation and oxidative stress in RAW 264.7 macrophages by suppressing the NF-B, MAPKs and PI3K/Akt pathways and activating Nrf2/HO-1 signaling, Int. J. Mol. Med. 41 (1) (2018) 264–274. [28] W.L. Li, H.W. Xin, M.W. Su, Inhibitory effects of continuous ingestion of Schisandrin A on CYP3A in the rat, Basic Clin. Pharmacol. Toxicol. 110 (2) (2012) 187–192.
Author contributions D. X. and J. L. designed the experiments, analyzed the data, prepared the figures and wrote the manuscript. D. X., J. L., H. M., W. G. and J. W. carried out experiments. X. K., Y. L., Q. G., Y. C. and J. C. participated in the feeding of mice, sample collection, and preparation in the experimental process. S. F. supervised the whole project and contributed to the editing of the manuscript. Declaration of Competing Interest There are no conflicts to declare. Acknowledgements This work was funded by the National Natural Science Foundation of China ((project No. 31602020, 31672509 and 31873004), Jilin Scientific and Technological Development Program (Project No. 20190103021JH), JLU Science and Technology Innovative Research Team. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.105983. 9
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(6016) (2011) 456–461. [38] 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 (12) (2014) 2694–2701. [39] H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B.B. Aggarwal, Y. Kondo, Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways, Mol. Pharmacol. 72 (1) (2007) 29–39. [40] J.W. Lee, H. Nam, L.E. Kim, Y. Jeon, H. Min, S. Ha, Y. Lee, S.Y. Kim, S.J. Lee, E.K. Kim, S.W. Yu, TLR4 (toll-like receptor 4) activation suppresses autophagy through inhibition of FOXO3 and impairs phagocytic capacity of microglia, Autophagy (2018). [41] Y. He, H. She, T. Zhang, H. Xu, L. Cheng, M. Yepes, Y. Zhao, Z. Mao, p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1, J. Cell Biol. 217 (1) (2018) 315–328. [42] I.Y. Lin, M.H. Pan, C.S. Lai, T.T. Lin, C.T. Chen, T.S. Chung, C.L. Chen, C.H. Lin, W.C. Chuang, M.C. Lee, C.C. Lin, N. Ma, CCM111, the water extract of Antrodia cinnamomea, regulates immune-related activity through STAT3 and NF-kappaB pathways, Sci. Rep. 7 (1) (2017) 4862. [43] C.Y. Wang, M.W. Mayo, A.S. Baldwin Jr., TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB, Science 274 (5288) (1996) 784–787. [44] F. Pei, H.S. Wang, Z. Chen, L. Zhang, Autophagy regulates odontoblast differentiation by suppressing NF-kappaB activation in an inflammatory environment, Cell Death Dis. 7 (2016) e2122.
[29] Y. Zhi, Y. Jin, L. Pan, A. Zhang, F. Liu, Schisandrin A ameliorates MPTP-induced Parkinson's disease in a mouse model via regulation of brain autophagy, Arch. Pharm. Res. (2019). [30] J.J. Wang, Z.K. Wei, X. Zhang, Y.N. Wang, Y.H. Fu, Z.T. Yang, Butyrate protects against disruption of the blood-milk barrier and moderates inflammatory responses in a model of mastitis induced by lipopolysaccharide, Br. J. Pharmacol. 174 (21) (2017) 3811–3822. [31] G. Shao, Y. Tian, H. Wang, F. Liu, G. Xie, Protective effects of melatonin on lipopolysaccharide-induced mastitis in mice, Int. Immunopharmacol. 29 (2) (2015) 263–268. [32] Q. Gong, Y. Li, H. Ma, W. Guo, X. Kan, D. Xu, J. Liu, S. Fu, Peiminine protects against lipopolysaccharide-induced mastitis by inhibiting the AKT/NF-kappaB, ERK1/2 and p38 signaling pathways, Int. J. Mol. Sci. 19 (9) (2018). [33] I.L. Chapple, J.B. Matthews, The role of reactive oxygen and antioxidant species in periodontal tissue destruction, Periodontol 2000 (43) (2007) 160–232. [34] C.H. Jung, S.H. Ro, J. Cao, N.M. Otto, D.H. Kim, mTOR regulation of autophagy, FEBS Lett. 584 (7) (2010) 1287–1295. [35] Y. Liu, H. Yu, X. Zhang, Y. Wang, Z. Song, J. Zhao, H. Shi, R. Li, Y. Wang, L.W. Zhang, The protective role of autophagy in nephrotoxicity induced by bismuth nanoparticles through AMPK/mTOR pathway, Nanotoxicology (2018) 1–16. [36] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2) (2011) 132–141. [37] D.F. Egan, D.B. Shackelford, M.M. Mihaylova, S. Gelino, R.A. Kohnz, W. Mair, D.S. Vasquez, A. Joshi, D.M. Gwinn, R. Taylor, J.M. Asara, J. Fitzpatrick, A. Dillin, B. Viollet, M. Kundu, M. Hansen, R.J. Shaw, Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy, Science 331
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Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy Dianwen Xu1, Juxiong Liu1, He Ma, Wenjin Guo, Jiaxin Wang, Xingchi Kan, Yanwei Li, ⁎ Qian Gong, Yu Cao, Ji Cheng, Shoupeng Fu College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Schisandrin A Mastitis Nrf2 Autophagy mTOR AMPK-ULK1
Schisandrin A (Sch A), a dibenzocyclooctadiene lignan extracted from Schisandra chinensis (Turcz.) Baill., has anti-oxidant and anti-inflammatory effects, but the effect on masitits has not been studied. Therefore, we investigated the effect of Sch A in cell and mouse models of lipopolysaccharide (LPS)-induced mastitis. Studies in vivo showed that Sch A reduced LPS-induced mammary injury and the production of pro-inflammatory mediators. Sch A also decreased the levels of pro-inflammatory mediators and activated nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway in mouse mammary epithelial cells (mMECs). The Nrf2 inhibitor partially abrogated the downregulation of Sch A on LPS-induced inflammatory response. In addition, LPS stimulation suppressed autophagy, while both Sch A and the autophagy inducer rapamycin activated autophagy in mMECs, which down-regulated inflammatory response. Sch A also restrained LPS-induced phosphorylation of mammalian target of rapamycin (mTOR) and activated AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1). In summary, these results suggest that Sch A exerts protective effects in LPS-induced mastitis models by activating Nrf2 signaling pathway and inducing autophagy and the autophagy is initiated by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway.
1. Introduction Mastitis refers to the inflammation of mammary gland that not only affects the health benefits of breastfeeding in lactating women but is also one of the most frequent and costly diseases in the dairy industry [1–4]. Mastitis is mainly caused by bacterial infections, including Gram-negative bacteria [5], such as Escherichia coli, which has been documented as one of the major etiological agents of mastitis [6]. It is typically characterized by an infiltration of somatic cells, mainly polymorphonuclear cells, into mammary tissues [7]. Although the pathogenesis of mastitis has not been fully elucidated, studies have shown that oxidative stress and autophagy may be involved [7,8]. The inflammatory response is associated with oxidative stress [9]. Mastitis is accompanied by a systemic oxidative stress response [10]. Oxidative stress process can kill pathogenic microorganisms, but it can also destroy various biomolecules and cause damage to mammary tissues [11]. Therefore, the body has developed various antioxidant mechanisms to counterbalance oxidative stress, such as nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway and macroautophagy
(hereafter referred to as autophagy) [12–15]. The Nrf2 pathway is a major regulator of cellular oxidative stress and protects cells from oxidative stress damage [16]. Under normal physiological conditions, the transcription factor Nrf2 interacts with its inhibitory protein Kelchlike ECH-associated protein 1 (Keap-1) and located in the cytoplasm. Upon exposure to stressors, Nrf2 is disconnected from Keap-1 and translocates into the nucleus to transactivate the transcription of antioxidant genes and cytoprotective genes, such as autophagy-related genes, thereby promoting cell survival [17–19]. However, in Escherichia coli-induced acute mastitis, the body's oxidative stress levels may overwhelm the antioxidant defense mechanisms, thereby aggravating inflammation and causing extensive mammary damage [11]. In addition, studies have found that autophagy, as a lysosomal-dependent protein degradation pathway [20], can inhibit inflammation by limiting activation of the inflammasomes and suppressing the secretion of proinflammatory cytokines [21,22]. Moreover, impaired autophagy is involved in the susceptibility of cows to mastitis [8], suggesting that autophagy may have an important effect on mastitis. Therefore, therapeutic strategies for regulating the body's antioxidant capacity and
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Corresponding author. E-mail address: [email protected] (S. Fu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.intimp.2019.105983 Received 16 August 2019; Received in revised form 11 October 2019; Accepted 14 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Dianwen Xu, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105983
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previously described [32]. Briefly, mammary tissues were homogenized with an appropriate amount of HEPES (N-2-hydroxyethylpiperazine-Nethane-sulphonicacid) solution and the supernatant was isolated for ELISA after ultracentrifugation. Subsequently, the precipitate was homogenized again with 0.5% CTAC (Cetyltrimethylammonium chloride) equivalent to HEPES and the supernatant was collected for MPO activity assay after centrifugation. 75 μL of sample was added into a 96-well plate after 10-fold dilution with 5% CTAC and each sample had three replicates. After that, 75 μL of substrate was added into the plate, and 2–3 min later, the reaction was terminated with 100 μL of 2 mol/L sulfuric acid. Finally, the absorbance at 450 nm was determined.
autophagy progression may be effective in treating mastitis. Schisandrin A (Sch A) is a biologically active dibenzocyclooctadiene lignan extracted from traditional Chinese herbal medicine Schisandra chinensis (Turcz.) Baill. [23]. A growing number of studies indicate that Sch A has anti-oxidant and anti-inflammatory effects [24–27]. However, whether Sch A can protect against lipopolysaccharide (LPS)-induced mastitis is unknown. Therefore, our study investigated the effect of Sch A in cell and mouse models of LPS-induced mastitis and its underlying mechanisms. 2. Materials and methods 2.1. Reagents
2.5. Determination of cytokine levels
Schisandrin A (Sch A, purity ≥ 98%) was bought from Shanghai YuanYe Biotechnology (Shanghai, China). Lipopolysaccharides from Escherichia coli O55:B5, all-trans-retinoic acid (ATRA) (purity ≥ 98%) and Compound C (purity ≥ 98%) were bought from Sigma-Aldrich (St Louis, MO, U SA). Rapamycin (Rap, purity = 99.30%) was obtained from Selleckchem (Houston, DE, USA). The primary antibodies against p-AMPKα (Thr172) (#2535), p-ERK1/2 (Thr202/Tyr204) (#9101), pAKT (Ser473) (#4070), p-mTOR (Ser2448) (#5536) and Histone H3 (#4499), AMPKα (#5832), mTOR (#2972), p-NF-κB p65 (#3033S), NF-κB p65 (#8242) and p-ULK1 (#5869T) were provided by Cell Signaling Technology (Beverly, MA, USA). Antibodies against ERK1/2 (16443-1-AP), AKT (10176-2-AP), ULK1 (20986-I-AP), LC3B (18725-IAP) and p62/SQSTM1 (18420-I-AP) were purchased from Proteintech (Wuhan, China). Antibodies against Nrf2 (NFE2L2) (ab31163), HO-1 (Heme Oxygenase-1) (ab68477), COX-2 (cyclooxygenase-2) (ab62331) and iNOS (inducible nitric oxide synthase) (ab15323) were purchased from Abcam (Cambridge, UK). β-Tubulin was bought from Beijing Ray Antibody Biotechnology (Beijing, China).
The content of cytokines TNF-α and IL-1β in tissues was measured by the enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Biolegend, San Diego, CA, USA) following the manufacturer’s instructions. 2.6. Cell culture Mouse mammary epithelial cells (mMECs) were obtained from the American Type Culture Collection (ATCC, ATCC®CRL-3063™) and maintained in DMEM medium (Gibco, Grand Island, NY 14072, USA) containing 10% fetal bovine serum (Clark Bioscience, Richmond, VA, USA) in a 37 °C incubator with 5% carbon dioxide. 2.7. CCK-8 assay The Cell Counting Kit-8 (CCK-8) (Saint-Bio, Shanghai, China) was applied to test whether Sch A affected cell viability. The mMECs were cultured in a 96-well plate for 12 h. After cultured in serum-free medium for 3 h, the cells were administrated with different molar concentrations of Sch A (40, 60, 80 and 100 μM) for 1 h, followed by exposure to LPS for 4 h. Subsequently, the cells in each well were incubated for 1 h after adding 10 μL of CCK-8 solution. Finally, the absorbance at 450 nm was determined.
2.2. Animal and treatment C57BL/6 mice (6–8 weeks old, 25–30 g) were provided by the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were kept in stainless, certified cages with good care conditions during experiments. In this study, all experiments were ratified by the Jilin University Institutional Animal Care and Use Committee and followed the guidelines (Protocol No. 2015047) established on 27 February 2015. One week after delivery, 30 female mice were stochastically assigned to 5 groups: Control group, Sch A group, LPS treatment group, LPS + Sch A group, and LPS + Dexamethasone (Dex) group with 6 mice in each group. Mice were administrated by intraperitoneal injection with 32 mg/kg of Sch A [28,29] or 5 mg/kg of Dex [30], respectively. One hour later, LPS was infused into the fourth pair of mammary glands via ducts. After 12 h, mice were administrated with 32 mg/kg of Sch A or 5 mg/kg of Dex, respectively, for another 12 h. Finally, the mice euthanized by cervical dislocation and the mammary glands were separated and stored as follow-up experimental samples.
2.8. Quantitative real-time PCR (qRT-PCR) Total RNA was separated from mMECs by using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and then was reverse-transcripted into cDNA by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Quantitative RT-PCR was performed with the SYBR Green QuantiTect RT-PCR Kit (Roche, South San Francisco, CA, USA). The primers were designed by Sangon Biotech (Shanghai, China) and the sequences are shown in Table 1. In this experiment, β-actin was selected as an internal reference gene and the relative mRNA levels of target genes were obtained by the 2−ΔΔCt method.
2.3. Assessment of histopathological changes
Table 1 The primer sequences of IL-1β, TNF-α, HO-1 and β-actin.
The mammary tissue blocks were fixed in 4% formaldehyde solution for 24 h, then embedded in paraffin, and finally made into 5 μm sections. Subsequently, to evaluate pathological changes, the sections were objected to staining with hematoxylin-eosin (H&E), observed and photographed under a light microscope. Finally, the pathological changes were determined with reference to the scores for injury degree as previously described [31]. 2.4. Determination of myeloperoxidase (MPO) activity The levels of MPO were determined according to the method 2
Gene
Sequences
Length (bp)
IL-1β
(F) 5′-GCAGAGCACAAGCCTGTCTTCC-3′ (R) 5′-ACCTGTCTTGGCCGAGGACTAAG-3′
198
TNF-α
(F) 5′-GCGACGTGGAACTGGCAGAAG-3′ (R) 5′-GCCACAAGCAGGAATGAGAAGAGG-3′
103
HO-1
(F) 5′-ACCGCCTTCCTGCTCAACATTG-3′ (R) 5′-CTCTGACGAAGTGACGCCATCTG-3′
104
β-actin
(F) 5′-ATCACTATTGGCAACGAGCGGTTC-3′ (R) 5′- CAGCACTGTGTTGGCATAGAGGTC-3′
156
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Fig. 1. Schisandrin A (Sch A) alleviates mammary injury in lipopolysaccharide (LPS)-induced mouse mastitis. Mice were pretreated with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treatment with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for H&E staining. (A) Control; (B) Sch A (32 mg/kg); (C) LPS (0.2 mg/kg); (D) LPS + Sch A (32 mg/kg); (E) LPS + Dex (5 mg/kg); (F) Mammary gland injury scores. Values are presented as mean ± SD (n = 6). The black arrows indicate the inflammatory cell infiltration in mammary tissues. Dex served as a positive control. Scale bar: 50 μm. ####P < 0.0001 vs. Control group and ****P < 0.0001 vs. LPS-treated group.
membrane (Millipore, Darmstadt, Germany) at 75 V for 1 h. The transferred membranes were blocked in TBS-T containing 5% skim milk for 2 h, followed by incubation overnight at 4 °C with each primary antibody in TBS-T containing 5% bovine serum albumin (Gentihold, Beijing, China). After washing five times with TBS-T, the PVDF membranes were co-incubated with the corresponding HRP-labeled secondary antibodies (1:3000 dilution) (Bosterbio, USA) at room temperature for 1 h. Finally, the immunoreactive blots were visualized with an enhanced chemiluminescent substrate (Beyotime, Shanghai, China). 2.10. Statistical analysis In this study, all data analysis work was performed using GraphPad Prism 6.0 software (La Jolla, CA, USA). The final results were presented in the form of mean ± SD. Statistical differences between groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons (P values < 0.05).
Fig. 2. Sch A down-regulates myeloperoxidase (MPO) activity in vivo. Mice were pretreated with Sch A (32 mg/kg) or Dex (5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for MPO activity assay. Values are presented as mean ± SD (n = 6). Dex served as a positive control. ####P < 0.0001 vs. Control group and ****P < 0.0001 vs. LPS-treated group.
3. Results 3.1. Sch A alleviates mammary injury in vivo
2.9. Western blotting To evaluate the effect of Sch A on mastitis, we constructed an LPSinduced mouse mastitis model. Mice were pretreated with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treating with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h and mammary glands were collected for H&E staining. The results showed that in comparison with the control group, the number of inflammatory cells infiltrating into the mammary gland cavity was remarkably increased by LPS treatment (Fig. 1A-C). However, treatment with Sch A (32 mg/ kg) or Dexamethasone (Dex, 5 mg/kg) significantly lowered LPS-induced infiltration of inflammatory cells (Fig. 1D-F). These results indicate that Sch A alleviates mammary injury in LPS-induced mouse
To obtain the total protein, homogenized mouse mammary tissues and treated mMECs were lysed by the addition of lysis buffer (Beyotime, Shanghai, China), followed by centrifugation for 15 min at 12,000g. The supernatants containing the total protein were collected. Similarly, cytoplasmic and nuclear proteins were obtained by applying the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Subsequently, the protein concentrations were determined by using the BCA Protein Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Protein samples were loaded onto 6%-15% SDS-PAGE at 110 V for 90 min and then transferred onto a PVDF 3
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Fig. 3. Sch A reduces the production of pro-inflammatory mediators in vivo. Mice were pretreated with Sch A (32 mg/kg) or Dex (5 mg/kg) for 1 h, and then were administrated with LPS (0.2 mg/kg) for 12 h followed by treatment with Sch A (32 mg/kg) or Dex (5 mg/kg) for another 12 h. Dex served as a positive control. (A-B) The protein levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). (C) The protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were determined by western blotting. (D-E) Bar graphs represent quantitative results for corresponding protein bands. Values are presented as mean ± SD (n = 6). ####P < 0.0001 vs. Control group; ****P < 0.0001 vs. LPS-treated group.
3.4. Sch A inhibits LPS-induced inflammatory response in mMECs
mastitis.
Before evaluating the role of Sch A, the cytotoxicity was determined by CCK-8 assay. The result showed that Sch A at concentrations of 40, 60, 80 and 100 μM did not alter the cell viability with or without LPS stimulation (5 μg/mL) (Fig. 4A). Subsequently, the anti-inflammatory effect of Sch A was evaluated. The qRT-PCR analysis showed that stimulation with LPS in mMECs induced over-expression of IL-1β and TNFα mRNA, which was reduced by Sch A at 10, 20 and 40 μM (Fig. 4B and C). Similarly, LPS treatment remarkably elevated the expressions of COX-2 and iNOS, which also was reduced by Sch A at 10, 20 and 40 μM (Fig. 4D-F). These results demonstrate that Sch A mitigates inflammatory response in LPS-stimulated mMECs.
3.2. Sch A down-regulates MPO activity in vivo At the same time, considering that myeloperoxidase (MPO) is an important indicator of neutrophil infiltration into tissues, oxidative stress and tissue damage [33], the levels of MPO in mammary tissues were determined. The results showed that in comparison with the control group, MPO activity was remarkably elevated by LPS treatment. However, treatment with Sch A (32 mg/kg) or Dexamethasone (Dex, 5 mg/kg) remarkably reduced MPO activity (Fig. 2). These results indicate that Sch A treatment significantly inhibits LPS-induced increase in MPO activity.
3.3. Sch A mitigates inflammatory response in vivo
3.5. The Nrf2 signaling pathway is involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs
The ELISA analysis showed that stimulation with LPS in mMECs increased the levels of IL-1β and TNF-α, which were reduced by Sch A treatment at 32 mg/kg (Fig. 3A and B). Similarly, LPS treatment remarkably elevated the protein expressions of COX-2 and iNOS, which were reduced by Sch A treatment (32 mg/kg) (Fig. 3C-E). Taken together, our data demonstrate that Sch A mitigates LPS-induced inflammatory response in vivo.
To evaluate the possibility that Nrf2 pathway was involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs, the mMECs were treated with Sch A (40 μM) at different time points (0, 1, 3, 6 and 12 h). Subsequently, the cells were collected for western blotting. The results showed that the levels of nuclear Nrf2 and Heme Oxygenase-1 (HO-1) were significantly increased with time (Fig. 5A-C). Although Sch A was proved to activate Nrf2 pathway, it was still 4
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Fig. 4. Sch A mitigates LPS-induced inflammatory response in vitro. (A) After serum starvation for 3 h, mouse mammary epithelial cells (mMECs) were administrated with different molar concentrations of Sch A (40, 60, 80 and 100 μM) for 1 h, followed by treatment with LPS for 4 h. Then, cell viability was determined by Cell Counting Kit-8 (CCK-8). (B-C) After serum starvation for 3 h, mMECs were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h, and then administrated with LPS for 4 h. Subsequently, the mRNA levels of IL-1β and TNF-α were detected by quantitative real-time PCR (qRT-PCR). (D) The protein levels of COX-2 and iNOS were determined by western blotting. (E-F) Bar graphs represent quantitative results for corresponding protein bands. Values are expressed as mean ± SD (n = 3). ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. LPS-treated group.
in LPS-stimulated mMECs. Although Sch A was proved to enhance autophagy, it was still unknown if autophagy was involved in the antiinflammatory effect of Sch A in LPS-stimulated mMECs. To evaluate the effect of autophagy, mMECs were treated with the autophagy inducer rapamycin (a specific mTOR inhibitor) for 2 h before LPS stimulation. After that, we observed that treatment with rapamycin increased the amount of LC3 II protein, lowered the amount of p62 protein (Fig. 6DF) and down-regulated the levels of IL-1β and TNF-α mRNA (Fig. 6I-J). In addition, both Sch A and rapamycin reduced phosphorylation of NFκB p65 (Fig. 6G-H). These results indicate that Sch A mitigates LPSinduced inflammatory response in mMECs, at least partially, due to autophagy induction.
unknown if Nrf2 pathway was involved in the roles of Sch A in LPSstimulated mMECs. Therefore, mMECs were treated with an Nrf2 inhibitor all-trans-retinoic acid (ATRA, 2 μM) for 6 h and then administrated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/ ml) for 4 h. After that, under the condition that Nrf2 was inhibited (Fig. 5D), Sch A still reduced the expression of TNF-α mRNA (Fig. 5F), but lost the inhibitory effect on the expression of IL-1β mRNA (Fig. 5E). These results indicate that Nrf2 inhibition partially abrogates the antiinflammatory effect of Sch A in LPS-stimulated mMECs, which means that Nrf2 pathway is partially responsible for the protective effect of Sch A in LPS-stimulated mMECs. 3.6. Sch A prevents LPS-induced inflammatory response by inducing autophagy in LPS-stimulated mMECs
3.7. Sch A induces autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs
To study if autophagy occurs in LPS-stimulated mMECs in the presence of Sch A, the autophagy makers LC3B (microtubule-associated protein 1 light chain 3 β) and p62 (SQSTM1/sequestosome 1) were determined by western blotting. Compared with the control group, LC3 II protein was decreased and p62 protein was increased after stimulation with LPS. However, treatment with Sch A at 10, 20 and 40 μM all elevated the amount of LC3 II protein and reduced the amount of p62 protein (Fig. 6A-C). The results indicate that Sch A induces autophagy
The mammalian target of rapamycin (mTOR) is a key negative regulator of autophagy activity [34]. To elucidate the molecular mechanism by which Sch A induced autophagy, we first evaluated the effect of Sch A on the phosphorylation status of mTOR. The results showed that LPS treatment elevated the level of p-mTOR, which was reduced by Sch A at 10, 20 and 40 μM (Fig. 7A and E). Subsequently, we determined the phosphorylation status of the upstream kinases protein 5
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Fig. 5. The nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway is involved in the anti-inflammatory effect of Sch A in LPS-stimulated mMECs. (A) mMECs were treated with 40 μM Sch A at different time points (0, 1, 3, 6 and 12 h). The protein levels of Nuclear-Nrf2 and Heme Oxygenase-1 (HO-1) were detected by western blotting. (B-C) Bar graphs represent quantitative results for corresponding protein bands. (D-E) mMECs were pretreated with a Nrf2 inhibitor all-transretinoic acid (ATRA, 2 μM) for 6 h and then treated with Sch A (40 μM) for 1 h , followed by exposure to LPS (5 μg/mL) for 4 h. The mRNA levels of IL-1β and TNF-α were determined by qRT-PCR. Values are presented as mean ± SD (n = 3). ##P < 0.01, ###P < 0.001 and ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. LPS-treated group; $P < 0.05 and $$$$P < 0.0001 vs. Sch A (40 μM) and LPS (5 μg/mL) co-treated group.
evaluated the effect of Sch A on the level of p-ULK1 (Ser555). The results showed that LPS treatment reduced the level of p-ULK1 (Ser555), which was elevated by Sch A at 10, 20 and 40 μM (Fig. 7F and G). To confirm that AMPK activation is responsible for the effect of Sch A on the level of p-ULK1 (Ser555), cells were pretreated with Compound C (CC, a potent inhibitor of AMPK, 10 μM) for 2 h and then administrated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/ml) for 4 h. The results showed that Sch A-induced phosphorylation of ULK1 at Ser555 was blocked by AMPK inhibitor (Fig. 7H-J). Collectively, these
kinase B (AKT), AMP-activated protein kinase (AMPK) and extracellular regulated protein kinase (ERK1/2). Among them, AMPK activation, AKT inhibition and ERK1/2 inhibition can all lead to inactivation of mTOR [35]. Our data showed that only the trends of p-AKT and pAMPK were consistent with that of p-mTOR (Fig. 7A, B, D and E), although the phosphorylation levels of the three kinases changed after treatment with Sch A (Fig. 7A-D). In addition, considering that AMPK can directly activate autophagy by phosphorylating unc-51 like kinase 1 (ULK1) at Ser 555 [36,37], we
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Fig. 6. Sch A prevents LPS-induced inflammatory response by inducing autophagy in LPS-stimulated mMECs. After serum starvation for 3 h, cells were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h or Rap (a specific inhibitor of mTOR, 200 nM) for 2 h, followed by treatment with LPS (5 μg/ mL) for 4 h. (A and D) The protein levels of LC3B and p62 were detected by western blotting. (B-C) and (E-F) Bar graphs represent quantitative results for corresponding protein bands. (G) The protein levels of p65 and p-p65 were detected by western blotting. (H) Bar graphs represent quantitative results for corresponding protein bands. (I-J) The mRNA levels of IL-1β and TNF-α were detected by qRT-PCR. Values are presented as mean ± SD (n = 3). ##P < 0.01, ### P < 0.001 and ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. LPS-treated group.
caused by pathogenic bacteria [1]. The typical pathological characteristic of mastitis is a large number of somatic cells, mainly neutrophils infiltrating into the mammary tissues [7]. In this study, we observed that Sch A treatment remarkably reduced LPS-induced infiltration of inflammatory cells into mammary tissues. Consistent with this notion, Sch A remarkably inhibited LPS-induced increase in MPO activity, a representative indicator of neutrophils infiltration into tissues, oxidative stress and tissue damage [33]. In addition, when exposed to inflammatory stimuli, such as LPS, cells produce excessive pro-inflammatory mediators such as IL-1β, TNF-α, COX-2 and iNOS, resulting in damage to mammary tissues [7]. In our study, both in vivo and in vitro experiments indicate that Sch A can mitigate LPS-induced mastitis. As mentioned earlier, because MPO activity can reflect the degree of oxidative stress, the results of MPO activity analysis indicate that Sch A may have a regulatory effect on oxidative stress in LPS-induced
results suggest that Sch A induces autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs. 4. Discussion Several pharmacological activities of Sch A have been demonstrated in previous studies, but whether Sch A has protective effects on mastitis is unknown. In this study, we showed that Sch A inhibits LPS-induced inflammatory response via activating Nrf2 signaling pathway and inducing autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway, which reduced mammary tissue damage and the production of pro-inflammatory mediators, ultimately inhibiting LPS-induced mouse mastitis. Mastitis refers to the inflammation of mammary gland mainly 7
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Fig. 7. Sch A activates autophagy by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway in LPS-stimulated mMECs. After serum starvation for 3 h, cells were administrated with different molar concentrations of Sch A (10, 20 and 40 μM) for 1 h, followed by treatment with LPS for 4 h. (A) The protein levels of p-mTOR, mTOR, p-AMPK, AMPK, p-ERK1/2, ERK1/2, p-AKT and AKT were determined by western blotting. (B-E) Bar graphs represent quantitative results for corresponding protein bands. (F) The protein levels of p-ULK1 and ULK1 were determined by western blotting. (G) Bar graphs represent quantitative results for corresponding protein bands. (H) mMECs were pretreated with Compound C (CC, 10 μM) for 2 h and then treated with Sch A (40 μM) for 1 h, followed by exposure to LPS (5 μg/mL) for 4 h. The protein levels of p-AMPK, AMPK, p-ULK1 and ULK1 were determined by western blotting. (I-J) Bar graphs represent quantitative results for corresponding protein bands. Values are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ####P < 0.0001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. LPS-treated group; $$$$P < 0.0001 vs. Sch A (40 μM) and LPS (5 μg/mL) co-treated group.
These results indicate that Nrf2 pathway partially contributes to the protective effect of Sch A in LPS-stimulated mMECs. However, because Nrf2 was not required for the inhibitory effect of Sch A on the expression of IL-1β mRNA, the underlying protective mechanisms of Sch A need to be further investigated. Autophagy is a process that relies on lysosomal degradation of proteins and organelles and is critical for cell survival and homeostasis maintenance [13]. Studies show that autophagy exerts the protective effects in several inflammatory diseases [38–41]. It is reported that impaired autophagy may be responsible for the increased susceptibility of cows to mastitis [8] and Sch A can induce autophagy in an acute lung injury model [26], which prompted us to investigate the possibility that Sch A suppresses LPS-induced mastitis by inducing autophagy. In this
mastitis. In response to sub-lethal stress such as oxidative stress, cells quickly adapt to adverse conditions through various stress response pathways to protect themselves from damage [12]. Among them, the transcription factor Nrf2 (nuclear factor-E2 associated factor 2) can transactivate the expression of antioxidant enzymes, such as Heme Oxygenase-1 (HO-1) [17]. These antioxidant enzymes can scavenge excess reactive oxygen species, thereby inhibiting the inflammatory response [38]. Sch A can activate Nrf2 signaling pathway in LPS-stimulated RAW 264.7 macrophages [27], consistently, our data suggest that Sch A can activate Nrf2 signaling pathway and induce HO-1 expression in mMECs. In addition, we observed that under the condition that Nrf2 was inhibited, Sch A still reduced the expression of TNF-α mRNA, but lost the inhibitory effect on the expression of IL-1β mRNA.
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study, for the first time, we showed that LPS stimulation suppresses autophagy [40,41] and Sch A treatment induces a protective autophagic process to inhibit LPS-induced mouse mammary epithelial inflammation. NF-κB is an important nuclear transcription factor that can transactivate the transcription of many pro-inflammatory genes [42]. LPS stimulation can induce NF-κB activation [43] and autophagy induction can suppress NF-κB activation by degrading p-NF-κB [44]. We investigated the relationship between autophagy induction and NF-κB down-regulation. In our study, when autophagy was induced, the level of p-NF-κB p65 was significantly reduced. This study provides a perspective to explain the possible mechanism of the protective effect of Sch A in LPS-induced mastitis, in which autophagy may play an essential role. Furthermore, we further investigated the possible mechanism of autophagy induction by Sch A. The kinase mTOR is the pivotal negative regulator of autophagy, and its activity is regulated by some upstream signaling pathways [38]. Previous studies have shown that the upstream signaling pathways involved in the regulation of mTOR activity mainly include AKT, AMPK and ERK1/2, among which AKT inhibition, ERK1/2 inhibition, and AMPK activation can all initiate autophagy [35]. Our results showed that only the trends of p-AKT and p-AMPK were consistent with that of p-mTOR, although the phosphorylation levels of the three kinases changed after treatment with Sch A. In addition, studies found that AMPK can directly activate autophagy by phosphorylating ULK1 at multiple sites, including Ser555 [36,37]. Consistent with this finding, AMPK activation is responsible for Sch Ainduced phosphorylation of ULK1 at Ser555. These results indicate that Sch A treatment inactivates mTOR signaling pathway and activates AMPK-ULK1 signaling pathway. These, in turn, promote autophagy initiation and mitigate LPS-induced inflammatory response. In summary, our results demonstrate that Sch A can attenuate mammary tissue damage and lower the production of pro-inflammatory mediators. These protective effects are mediated by activation of Nrf2 pathway and induction of autophagy. Moreover, the autophagy is initiated at least partially by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway. Taken together, our findings suggest that Sch A exerts a protective effect in cell and mouse models of LPS-induced mastitis and is promising for use in the treatment of mastitis.
References [1] A. Bradley, Bovine mastitis: an evolving disease, Vet. J. 164 (2) (2002) 116–128. [2] Y. Zhu, G. Liu, X. Du, Z. Shi, M. Jin, X. Sha, X. Li, Z. Wang, X. Li, Expression patterns of hepatic genes involved in lipid metabolism in cows with subclinical or clinical ketosis, J. Dairy Sci. (2018). [3] W. Yang, H. Zerbe, W. Petzl, R.M. Brunner, J. Gunther, C. Draing, S. von Aulock, H.J. Schuberth, H.M. Seyfert, Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NFkappaB in mammary epithelial cells and to quickly induce TNFalpha and interleukin-8 (CXCL8) expression in the udder, Mol. Immunol. 45 (5) (2008) 1385–1397. [4] M. Marin, R. Arroyo, I. Espinosa-Martos, L. Fernandez, J.M. Rodriguez, Identification of Emerging Human Mastitis Pathogens by MALDI-TOF and Assessment of Their Antibiotic Resistance Patterns, Front. Microbiol. 8 (2017) 1258. [5] J.L. Watts, Etiological agents of bovine mastitis, Vet. Microbiol. 16 (1) (1988) 41–66. [6] S. Passey, A. Bradley, H. Mellor, Escherichia coli isolated from bovine mastitis invade mammary cells by a modified endocytic pathway, Vet. Microbiol. 130 (1–2) (2008) 151–164. [7] X. Zhao, P. Lacasse, Mammary tissue damage during bovine mastitis: causes and control, J. Anim. Sci. 86 (13 Suppl) (2008) 57–65. [8] M. Sugimoto, Y. Sugimoto, Variant in the 5' untranslated region of insulin-like growth factor 1 receptor is associated with susceptibility to mastitis in cattle, G3 (Bethesda) 2 (9) (2012) 1077–1084. [9] Q. Chi, X. Chi, X. Hu, S. Wang, H. Zhang, S. Li, The effects of atmospheric hydrogen sulfide on peripheral blood lymphocytes of chickens: Perspectives on inflammation, oxidative stress and energy metabolism, Environ. Res. 167 (2018) 1–6. [10] R. Turk, C. Piras, M. Kovacic, M. Samardzija, H. Ahmed, M. De Canio, A. Urbani, Z.F. Mestric, A. Soggiu, L. Bonizzi, P. Roncada, Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis, J. Proteomics 75 (14) (2012) 4412–4428. [11] K. Lauzon, X. Zhao, A. Bouetard, L. Delbecchi, B. Paquette, P. Lacasse, Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage, J. Dairy Sci. 88 (12) (2005) 4295–4303. [12] G. Kroemer, G. Marino, B. Levine, Autophagy and the integrated stress response, Mol. Cell 40 (2) (2010) 280–293. [13] K. Taguchi, N. Fujikawa, M. Komatsu, T. Ishii, M. Unno, T. Akaike, H. Motohashi, M. Yamamoto, Keap1 degradation by autophagy for the maintenance of redox homeostasis, Proc. Natl. Acad. Sci. U S A 109 (34) (2012) 13561–13566. [14] R. Sumpter Jr., B. Levine, Autophagy and innate immunity: triggering, targeting and tuning, Semin. Cell Dev. Biol. 21 (7) (2010) 699–711. [15] M. Battino, F. Giampieri, F. Pistollato, A. Sureda, M.R. de Oliveira, V. Pittala, F. Fallarino, S.F. Nabavi, A.G. Atanasov, S.M. Nabavi, Nrf2 as regulator of innate immunity: A molecular Swiss army knife!, Biotechnol. Adv. 36 (2) (2018) 358–370. [16] K. Takaya, T. Suzuki, H. Motohashi, K. Onodera, S. Satomi, T.W. Kensler, M. Yamamoto, Validation of the multiple sensor mechanism of the Keap1-Nrf2 system, Free Radical. Bio Med. 53 (4) (2012) 817–827. [17] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [18] M. Pajares, N. Jimenez-Moreno, A.J. Garcia-Yague, M. Escoll, M.L. de Ceballos, F. Van Leuven, A. Rabano, M. Yamamoto, A.I. Rojo, A. Cuadrado, Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes, Autophagy 12 (10) (2016) 1902–1916. [19] S. Dayalan Naidu, D. Dikovskaya, E. Gaurilcikaite, E.V. Knatko, Z.R. Healy, H. Mohan, G. Koh, A. Laurell, G. Ball, D. Olagnier, L. de la Vega, I.G. Ganley, P. Talalay, A.T. Dinkova-Kostova, Transcription factors NRF2 and HSF1 have opposing functions in autophagy, Sci. Rep. 7 (1) (2017) 11023. [20] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (1) (2008) 27–42. [21] M. Takahama, S. Akira, T. Saitoh, Autophagy limits activation of the inflammasomes, Immunol. Rev. 281 (1) (2018) 62–73. [22] T. Monkkonen, J. Debnath, Inflammatory signaling cascades and autophagy in cancer, Autophagy 14 (2) (2018) 190–198. [23] A. Szopa, R. Ekiert, H. Ekiert, Current knowledge of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) as a medicinal plant species: a review on the bioactive components, pharmacological properties, analytical and biotechnological studies, Phytochem. Rev. 16 (2) (2017) 195–218. [24] F. Song, K. Zeng, L. Liao, Q. Yu, P. Tu, X. Wang, Schizandrin A Inhibits microgliamediated neuroninflammation through inhibiting TRAF6-NF-kappaB and Jak2Stat3 signaling pathways, PLoS One 11 (2) (2016) e0149991. [25] Y.H. Choi, Schizandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells, Biomed. Pharmacother. 106 (2018) 902–909. [26] Y. Lu, W.J. Wang, Y.Z. Song, Z.Q. Liang, The protective mechanism of schisandrin A in d-galactosamine-induced acute liver injury through activation of autophagy, Pharm. Biol. 52 (10) (2014) 1302–1307. [27] D.H. Kwon, H.J. Cha, E.O. Choi, S.H. Leem, G.Y. Kim, S.K. Moon, Y.C. Chang, S.J. Yun, H.J. Hwang, B.W. Kim, W.J. Kim, Y.H. Choi, Schisandrin A suppresses lipopolysaccharide-induced inflammation and oxidative stress in RAW 264.7 macrophages by suppressing the NF-B, MAPKs and PI3K/Akt pathways and activating Nrf2/HO-1 signaling, Int. J. Mol. Med. 41 (1) (2018) 264–274. [28] W.L. Li, H.W. Xin, M.W. Su, Inhibitory effects of continuous ingestion of Schisandrin A on CYP3A in the rat, Basic Clin. Pharmacol. Toxicol. 110 (2) (2012) 187–192.
Author contributions D. X. and J. L. designed the experiments, analyzed the data, prepared the figures and wrote the manuscript. D. X., J. L., H. M., W. G. and J. W. carried out experiments. X. K., Y. L., Q. G., Y. C. and J. C. participated in the feeding of mice, sample collection, and preparation in the experimental process. S. F. supervised the whole project and contributed to the editing of the manuscript. Declaration of Competing Interest There are no conflicts to declare. Acknowledgements This work was funded by the National Natural Science Foundation of China ((project No. 31602020, 31672509 and 31873004), Jilin Scientific and Technological Development Program (Project No. 20190103021JH), JLU Science and Technology Innovative Research Team. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.105983. 9
International Immunopharmacology xxx (xxxx) xxxx
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(6016) (2011) 456–461. [38] 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 (12) (2014) 2694–2701. [39] H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B.B. Aggarwal, Y. Kondo, Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways, Mol. Pharmacol. 72 (1) (2007) 29–39. [40] J.W. Lee, H. Nam, L.E. Kim, Y. Jeon, H. Min, S. Ha, Y. Lee, S.Y. Kim, S.J. Lee, E.K. Kim, S.W. Yu, TLR4 (toll-like receptor 4) activation suppresses autophagy through inhibition of FOXO3 and impairs phagocytic capacity of microglia, Autophagy (2018). [41] Y. He, H. She, T. Zhang, H. Xu, L. Cheng, M. Yepes, Y. Zhao, Z. Mao, p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1, J. Cell Biol. 217 (1) (2018) 315–328. [42] I.Y. Lin, M.H. Pan, C.S. Lai, T.T. Lin, C.T. Chen, T.S. Chung, C.L. Chen, C.H. Lin, W.C. Chuang, M.C. Lee, C.C. Lin, N. Ma, CCM111, the water extract of Antrodia cinnamomea, regulates immune-related activity through STAT3 and NF-kappaB pathways, Sci. Rep. 7 (1) (2017) 4862. [43] C.Y. Wang, M.W. Mayo, A.S. Baldwin Jr., TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB, Science 274 (5288) (1996) 784–787. [44] F. Pei, H.S. Wang, Z. Chen, L. Zhang, Autophagy regulates odontoblast differentiation by suppressing NF-kappaB activation in an inflammatory environment, Cell Death Dis. 7 (2016) e2122.
[29] Y. Zhi, Y. Jin, L. Pan, A. Zhang, F. Liu, Schisandrin A ameliorates MPTP-induced Parkinson's disease in a mouse model via regulation of brain autophagy, Arch. Pharm. Res. (2019). [30] J.J. Wang, Z.K. Wei, X. Zhang, Y.N. Wang, Y.H. Fu, Z.T. Yang, Butyrate protects against disruption of the blood-milk barrier and moderates inflammatory responses in a model of mastitis induced by lipopolysaccharide, Br. J. Pharmacol. 174 (21) (2017) 3811–3822. [31] G. Shao, Y. Tian, H. Wang, F. Liu, G. Xie, Protective effects of melatonin on lipopolysaccharide-induced mastitis in mice, Int. Immunopharmacol. 29 (2) (2015) 263–268. [32] Q. Gong, Y. Li, H. Ma, W. Guo, X. Kan, D. Xu, J. Liu, S. Fu, Peiminine protects against lipopolysaccharide-induced mastitis by inhibiting the AKT/NF-kappaB, ERK1/2 and p38 signaling pathways, Int. J. Mol. Sci. 19 (9) (2018). [33] I.L. Chapple, J.B. Matthews, The role of reactive oxygen and antioxidant species in periodontal tissue destruction, Periodontol 2000 (43) (2007) 160–232. [34] C.H. Jung, S.H. Ro, J. Cao, N.M. Otto, D.H. Kim, mTOR regulation of autophagy, FEBS Lett. 584 (7) (2010) 1287–1295. [35] Y. Liu, H. Yu, X. Zhang, Y. Wang, Z. Song, J. Zhao, H. Shi, R. Li, Y. Wang, L.W. Zhang, The protective role of autophagy in nephrotoxicity induced by bismuth nanoparticles through AMPK/mTOR pathway, Nanotoxicology (2018) 1–16. [36] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2) (2011) 132–141. [37] D.F. Egan, D.B. Shackelford, M.M. Mihaylova, S. Gelino, R.A. Kohnz, W. Mair, D.S. Vasquez, A. Joshi, D.M. Gwinn, R. Taylor, J.M. Asara, J. Fitzpatrick, A. Dillin, B. Viollet, M. Kundu, M. Hansen, R.J. Shaw, Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy, Science 331
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