The intestinal mucosa is one of the main points of contact between the body and the external environment [1
]. Small intestinal epithelial cells are the first physiological line of defense in the mucosa, which selectively absorbs nutrients and protects against pathogenic invasions from the external environment [2
]. Foreign pathogens and environmental changes can easily result in gastrointestinal diseases, such as infectious enterocolitis, irritable bowel syndrome, and inflammatory bowel disease [3
]. Therefore, it is essential to maintain proper intestinal epithelial structure and function.
Oxidative stress, arising from the excessive generation of reactive oxygen species (ROS) and imbalances in the antioxidant system, can damage multiple cellular components through DNA hydroxylation, protein denaturation, lipid peroxidation, and membrane rupture. This outcome leads to apoptosis and other modes of cell death [4
]. Given the small intestine of calf gastrointestinal hypoplasia, oxidative stress can disrupt the integrity and functioning of calf intestinal epithelial cells, thereby affecting absorption and allowing pathogen invasion and disease [6
]. Oxidative stress plays a major role in the pathogenesis of a variety of disorders, including intestinal diseases [7
]. Cellular defenses against ROS damage include both enzymatic and nonenzymatic antioxidant systems, which act to reestablish or maintain redox homeostasis [11
]. Enzymatic antioxidant systems mainly involve enzymes that can scavenge or convert free radicals and ROS, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), while nonenzymatic antioxidant systems involve synthetic and plant-extracted compounds [12
]. Importantly, the activation of enzymatic antioxidant systems often requires an activated nonenzymatic antioxidant system as a precursor. Therefore, identifying safe and effective nonenzymatic antioxidants is essential to preventing and alleviating the damage caused by oxidative stress.
The nuclear factor erythroid 2-related factor 2 (NFE2L2)-antioxidant responsive element (ARE) signaling pathway plays a vital role in the cellular responses to oxidative stress-induced injuries [13
]. NFE2L2 is a transcription factor that is highly sensitive to oxidative stress. It binds to AREs on the chromatin and promotes the transcription of a wide variety of antioxidant enzymes [14
]. Normally, NFE2L2 is sequestered in the cytoplasm by Kelch-like ECH associated protein 1 (KEAP1). However, upon exposure to oxidative stress or chemopreventive compounds, NFE2L2 dissociates from KEAP1 and then translocate to the nucleus, where it heterodimerizes with its obligatory partner MAF bZIP transcription factor, binds AREs on the chromatin, and then activates ARE-mediated phase II detoxification by increasing the expression of a wide variety of antioxidant enzymes. This process includes heme oxygenase-1 (HMOX1), NAD(P)H quinone dehydrogenase 1 (NQO1), SOD, CAT, and GSH-Px [15
Astragaloside IV (ASIV; Figure 1
) is one of the main active components of the traditional Chinese medicinal plant Astragalus membranaceus
. ASIV has several pharmacological properties, including anti-inflammatory, antiapoptotic, and antioxidative effects [17
]. Recent studies have reported the strong antioxidative effects of ASIV, which can remove ROS and decrease lipid peroxidation [20
]. However, the mechanisms by which ASIV ameliorates oxidative stress remain largely unknown. In this study, we investigated the protective role of ASIV against H2
-induced oxidative stress in calf small intestine epithelial cells, as well as its mechanism of action.
The intestinal mucosa is one of the main barriers between the body and the external environment, and it functions by selectively absorbing nutrients and preventing invasion from the external environment. The integrity of the small intestine is essential for proper nutrient absorption, gut homeostasis, and organismal growth and health [6
]. The production of ROS during oxidative stress creates an imbalance between the ability of intestinal epithelia to counteract to detoxify the harmful effects of ROS with intrinsic antioxidants and other protective mechanisms [22
]. Furthermore, oxidative stress leads to immune system dysfunction and affects the digestion, absorption, and transformation of nutrients. Therefore, it is a critical factor in intestinal disease pathogenesis. ASIV, an active ingredient isolated from Astragalus membranaceus
, has demonstrated antioxidative and protective effects on gastrointestinal health [23
Numerous studies have demonstrated that H2
induces oxidative stress in various cell types, making it a classic in vitro model of oxidative stress [25
]. ROS accumulation can cause severe cellular injury and result in decreased cell repair and regeneration [28
]. In this study, we found that the viability of calf small intestine epithelia cells exposed to 350 μM H2
decreased by up to 41%, whereas ASIV pretreatment significantly inhibited H2
-induced cytotoxicity and increased cell viability. Importantly, ASIV was not toxic to cells. In addition, ASIV pretreatment decreased H2
-induced membrane damage and LDH release. These results indicated that ASIV had cytoprotective effects on calf intestine epithelia cells. Oxidative stress occurs when redox homeostasis is disrupted, and the cellular ROS overproduction results from the imbalance between oxidants and antioxidants [29
]. SOD, GSH-Px, and CAT are key antioxidative enzymes in the defense against oxidative stress, and they act by scavenging ROS [30
]. SOD and GSH-Px play an essential role in scavenging free radicals, and CAT can catalyze the decomposition of H2
to produce molecular oxygen (O2
) and H2
]. Increased SOD, GSH-Px, and CAT levels contribute to the resolution of oxidative stress, and decreased levels exacerbate oxidative damage [22
]. MDA levels also reflect the oxidative damage in calf small intestine epithelia cells, and it is crucial to protect cells by reducing ROS and MDA formation and enhance the SOD, GSH-Px, and CAT levels. In our study, the exposure of calf small intestine epithelia cells to H2
induced notable decreases in the SOD, GSH-Px, and CAT levels, in addition to marked increases in ROS and MDA production. However, ASIV pretreatment significantly reversed these effects, consistent with previous reports [33
]. These results may have been because the accumulation of intracellular ROS caused protein denaturation, which led to glycosylation and decreased antioxidative enzyme activity [35
]. ASIV can enhance the antioxidant capacity of cells by increasing the levels of antioxidant enzymes, thereby inhibiting cell damage caused by the accumulation of intracellular ROS. Oxidative stress usually induces apoptosis [36
], and our results indicated that H2
-induced apoptosis in calf small intestine epithelial cells was notably reduced by pretreatment with ASIV, reinforcing the notion that ASIV prevents H2
-induced oxidative stress by increasing the endogenous antioxidant levels.
Activation of the antioxidant system is crucial to preventing oxidative damage. NFE2L2 is a target of exogenous toxic substances and oxidative stress, and it plays a vital role in the presence of these insults [37
]. Normally, NFE2L2 binds to KEAP1 in the cytoplasm, which prevents its translocation to the nucleus and inhibits the transcription of a series of cytoprotective genes [38
]. Under oxidative conditions, NFE2L2 dissociates from KEAP1 and translocates to the nucleus, where it binds to AREs and promotes the expression of numerous phase II enzymes, including HMOX1 and NQO1 [39
]. Studies have demonstrated that the NFE2L2-ARE signal pathway could activate antioxidant enzymes, such as SOD, CAT, and GSH-Px, thereby enhancing the ability of small intestine epithelial cells to clean up ROS, protecting against oxidative damage [40
] In this study, ASIV pretreatment increased the mRNA and protein expression levels of NFE2L2, HMOX1, and NQO1. To further investigate the role of NFE2L2-ARE signaling after H2
exposure, we used ML385, an inhibitor of NFE2L2, and found that ML385 markedly downregulated the expression levels of these proteins in the presence of ASIV. Moreover, the ASIV pretreatment enhanced the nuclear fluorescence intensity of NFE2L2, HMOX1, and NQO1. Collectively, these results indicate that the protective effects of ASIV against H2
-induced calf small intestine epithelial cell oxidative damage may be attributed to the upregulation of HMOX1 and NQO1 via the NFE2L2-ARE signaling pathway, consistent with previous reports [22
In conclusion, the present study indicates that ASIV treatment effectively protects against H2O2-induced oxidative damage, thereby inhibiting ROS overproduction, MDA formation, and apoptosis by upregulating SOD, GSH-Px, and CAT levels. The mechanisms underlying the protective effects of ASIV may be related to the activation of the NFE2L2-ARE signal pathway. More efforts are required to reveal the functions and mechanisms of ASIV on intestinal disease using further in vitro and in vivo studies.
4. Materials and Methods
4.1. Chemicals and Reagents
Astragaloside IV (> 98%) was purchased from Nanjing Spring & Autumn Biological Engineering (Nanjing, China) and stored in the dark at −20°C and then dissolved in dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China). It was also diluted with complete medium prior to experimentation. H2O2 and t-BHQ were obtained from Sigma-Aldrich (St. Louis, MO, USA). Penicillin and streptomycin, Fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). Primary antibodies used targeted NFE2L2 (16396-1-AP, Proteintech, Chicago, IL, USA), NQO1 (11451-1-AP, Proteintech, Chicago, IL, USA), HMOX1 (ab13248, Abcam, Cambridge, UK), and β-actin (GB12001, Servicebio, Wuhan, China). GSH-Px, SOD, CAT, T-AOC, and MDA test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
4.2. Cell Culture and Treatments
The Shandong Academy of Agricultural Sciences provided calf small intestine epithelial cells. Cells were cultured in DMEM/F-12 containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. At 80% confluence, the cells were treated with 0, 10, or 25 nM ASIV for 12 h before treatment with 350 μM H2O2 for 12 h. Positive control cells were treated with 25 nM t-BHQ.
4.3. Cell Viability and LDH Leakage Assays
Cell viability was measured using the cell counting kit 8 (CCK8) assay (MedChemExpress, Monmouth Junction, NJ, USA) following the manufacturer’s instructions. Cells (2 × 104 cells/well) were plated in 96-well plates and incubated for 24 h. Then they were incubated in H2O2 or ASIV for some time. The cells were then incubated in DMEM/F-12 containing 10% CCK8 reagent at 37 °C for 2 h. Absorbance at 450 nm was recorded with a microplate reader. LDH levels were measured by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
4.4. Measurement of Intracellular ROS Levels
Intracellular ROS levels were evaluated using the ROS detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Cells (1 × 106 cells/mL) were cultured in 6-well plates, pretreated with different concentrations of ASIV or 25 nM t-BHQ for 12 h at 37 °C, and then they were treated with 350 μM H2O2 for 12 h at 37 °C. The cells were washed thrice with phosphate-buffered saline (PBS), incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, and then washed thrice with serum-free DMEM/F-12. Fluorescence intensities were measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
4.5. Apoptosis Analysis
Apoptosis used the Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China), and was determined by flow cytometry. The cells were washed twice with ice-cold PBS, digested and separated with 0.25% trypsin at 37 °C for 3 min, and then neutralized the trypsin with DMEM/F-12 containing 10% FBS. The cells were collected and centrifuged at 1500 rpm/min for 5 min. Next, the cells were resuspended in 100 μL binding buffer containing 5 μL Annexin V-FITC and 5 μL PI and then incubated at 4°C in the dark for 20 min. Apoptosis detection was performed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
4.6. Measurement of the Intracellular GSH-Px, CAT, SOD, T-AOC, and MDA Levels
Cells (1 × 106/mL) were plated in 6-well plates and then treated with ASIV or t-BHQ for 12 h before exposure to 350 μM H2O2 for 12 h. The GSH-Px, CAT, SOD, T-AOC, and MDA levels were quantified using commercially available kits, according to the manufacturer’s instructions.
4.7. RNA Extraction and qRT-PCR Analysis
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China), following the manufacturers’ instructions. The reverse transcription process was as follows: 50 °C for 15 min, and 85 °C for 5 s. The primer sequences are shown in Table 1
, and they were synthesized by Sangon Biotech (Shanghai, China). We performed qRT-PCR on a LightCycler 480 (Roche Diagnostics, Burgess Hill, UK), using the ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) with the following conditions: stage 1, 95 °C for 30 s; stage 2, 40 cycles of 95 °C for 10 s and 60 °C for 30 s; and stage 3, 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. Relative gene expression levels were determined using the 2−ΔΔCT
4.8. NFE2L2, NQO1, and HMOX1 Immunofluorescence
Cells (1 × 106/mL) were cultured in 24-well plates, rinsed thrice with PBS, and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were washed again with PBS, followed by permeabilization for 20 min in 0.1% Triton X-100. After washing with PBS, the cells were blocked in 5% FBS for 30 min. The blocking solution was removed and replaced with primary antibodies against NFE2L2 (1:200), NQO1 (1:50), and HMOX1 (1:300) in 0.5% FBS. The cells were then incubated overnight at 4 °C and then washed thrice with PBS before incubation with goat anti-rabbit (AS00029, GenStar, Beijing, China) or goat anti-mouse (AS00071, GenStar, Beijing, China) secondary antibodies for 1 h. The cells were washed thrice in PBS, and nuclear counterstaining was performed by incubation with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Images were acquired using a BX51 fluorescence microscope (Olympus, Tokyo, Japan) and the Lecia Application Suite (Version 4.12.0, Germany) analysis software.
4.9. Western Blot Analysis
Cells were lysed in radioimmunoprecipitation buffer (G2002, Servicebio, Wuhan, China) containing protease and phosphatase inhibitors (G2007, Servicebio, Wuhan, China) for 30 min. Concentrations of protein were measured using a BCA protein assay kit (Beijing ComWin Biotech, Beijing, China). Equivalent amounts of proteins were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% (w/v) nonfat dry milk for 1 h and then incubation at 4 °C overnight with primary antibodies against NFE2L2 (1:1000), NQO1 (1:500), HMOX1 (1:250), and β-actin (1:1500). After washing thrice with 0.5% Tris-buffered saline/TWEEN (TBST), the membrane were incubated with horseradish peroxidase-conjugated goat anti-rabbit (GB23303, Servicebio, Wuhan, China) or goat anti-mouse antibodies (GB23301, Servicebio, Wuhan, China) for 2 h at room temperature. The bands were detected by an ECL (G2014, Servicebio, Wuhan, China), and the band intensities were quantified using the Alpha analysis software (alphaEaseFC, Alpha Innotech, Santa Clara, CA, USA).
4.10. Statistical Analysis
All data are presented as the mean ± standard error of the mean (SEM). Comparisons between different groups were analyzed using a one-way analysis of variance and Tukey′s post hoc test in SAS V8 (SAS Institute, Cary, NC, USA). Statistical significance was defined as p < 0.05 or p < 0.01.