Dihydroartemisinin ameliorates cerebral I/R injury in rats via regulating VWF and autophagy-mediated SIRT1/FOXO1 pathway

2.1 Animals and ethics statement

This study was carried out on newborn Sprague Dawley (SD) rats (10 days old) and adult male SD rats weighing 240–310 g (Charles River Laboratories, Beijing, China). The rats were housed in a specific pathogen-free environment and given a standard diet and drinking water ad libitum. Animal surgical procedures were approved by the Committee of Laboratory Animals of Nantong Rich Hospital (CIR20201006), and performed in accordance with the Guideline for the Care and Use of Laboratory Animals.

2.2 Cerebral I/R procedures and drug administration

All rats were randomly divided into three groups: Control group, I/R group, and I/R + DHA group. The cerebral I/R model was constructed using middle cerebral artery occlusion (MCAO), as previously reported [25]. Briefly, the rats in the I/R group and I/R + DHA group were anesthetized with pentobarbital sodium (45 mg/kg, P-010; Whitehouse Station, New Jersey, Merck, USA). Then, a midline incision was created to expose the right common carotid artery, internal carotid artery, and external carotid artery (ECA). After the ECA was separated, a nylon suture (0.26 mm in diameter) was inserted into the lumen of the ECA for 18–20 mm until blunted distal end met resistance. Two hours later, the incision was sutured when the suture was withdrawn. The rats in the Control group received identical surgical procedures aside from the occlusion. Subsequently, 15 min later, 0.5 mL dimethyl sulfoxide (DMSO, 10% v/v, D8418, Merck, USA) and DMSO-dissolved DHA (0.1 mg/kg, C₁₅H₂₄O₅, D140839, Aladdin, China) were used to treat rats in I/R group and I/R + DHA group, respectively, by tail vein injection [24]. Following 14 days of reperfusion, all animals were euthanized by anesthesia and cervical dislocation for histological analysis of brain tissues.

2.3 Histological analysis

To evaluate the neuroprotective effect of DHA on I/R rat brain, 2,3,5-triphenyltetrazolium chloride (TTC) staining was applied to calculate cerebral infarct volumes, as previously described [26]. The frozen brain tissues were cut into sections (5 μm) and stained with TTC solution (17779, Merck, USA) at 37°C in the dark for 30 min. Then, the TTC-stained sections were fixed with 4% paraformaldehyde (abs9179, Absin, China). Infarction areas, which are manifested by a lack of red staining on the sections, were quantified using Image J software (vision 1.8.0, National Institutes of Health, Bethesda, MD, USA).

For morphological evaluation, hematoxylin and eosin (HE) staining was carried out using HE Staining Kit (G1121, Solarbio, China). According to the protocols, the frozen sections were stained with hematoxylin at room temperature for 10 min followed by differentiation. Afterward, the sections were subjected to bluing treatment, then were stained with eosin for 2 min and washed with water. A microscope (CX23, Olympus, Japan) was finally used for observation under ×100 magnification.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was conducted using a TUNEL kit (MK1015, Wuhan Boster Biological Technology, Ltd). A microscope was applied to observe five randomly selected fields in the injury site of brain tissues in a blinded manner and determine the TUNEL-positive neurons.

2.4 Cell culture

BMVECs were isolated from newborn SD rats as previously described [6]. In brief, the sacrificed rats were scrubbed with 75% ethanol (E111991, Aladdin, China) and the cranial cavity was exposed. Next, the cerebral cortex was separated after removal of the meninges and blood vessels. The cortex was minced, and collagenase and dispase two-step combined dissociation (10269638001, Roche, Basel, Switzerland) was used to digest the tissues. By centrifugation at 1,000×g for 5 min at 4℃, BMVECs were aspirated and suspended with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, 11320033, Thermo Fisher, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS, 10091155, Thermo Fisher, USA) and basic fibroblast growth factor (70 ng/mL, TL-401, Wolcavi, China). Lastly, BMVECs were seeded (1 × 10⁵/cm²) in a gelatin-coated plastic dish at 37°C with 5% CO₂ for 2 days of subculture.

2.5 Oxygen–glucose deprivation/reoxygenation (OGD/R) treatment

The cultured BMVECs were treated with glucose-free medium (E600010-0500, Sangon Biotech, China) with 2% FBS in an incubator (37°C, 5% CO₂ and 95% N₂) as per guidance. Six hours later, the cells were transferred to the standard medium and incubated in a normoxic condition (25% O₂, 5% CO₂, and 70% N₂) for 4 h. BMVECs cultured in the normoxic condition were used as the control.

2.6 Cell transfection and DHA treatment

VWF-overexpressing plasmids (Bes-mR-053889) and control plasmids (NC) were purchased from BersinBio (Guangzhou, China). BMVECs were transfected with VWF-overexpressing plasmids or control plasmids by Lipofectamine 3000 (L3000008, Thermo Fisher, USA) according to the specification. After OGD/R treatment, the cells were incubated with 25 μM DHA for 24 h [23]. In the end, five groups were constructed: Control group, OGD/R group, OGD/R + DHA group, OGD/R + DHA + NC group, and OGD/R + DHA + VWF group. The cells from above groups were subsequently subjected to quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), cell function experiments, and Western blot.

2.7 RNA extraction and qRT-PCR

Total RNA from BMVECs with different treatments was isolated with Trizol (9108, Takara, Japan) according to the manufacturer’s instructions, and then was reverse-transcribed using cDNA Synthesis Kit (6130, Takara, Japan). After that, qRT-PCR was conducted on a Real-Time System (CFX96, Bio-Rad, Hercules, CA, USA) with Fast SYBR Green Master Mix (4385612, Thermo Fisher, USA). The gene-specific primer sequences were VWF (forward: 5′-ATGGCCCTTTCCTGACCTAC-3′; reverse: 5′-GGATTAGGGTTGGCCCTGAG-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, forward: 5′-TAATGCCGCCCCTTACCATC-3′; reverse: 5′-GGTGCAGCGATGCTTTACTT-3′). The conditions of PCR reaction were as follows: predenaturation at 95°C for 20 s and 40 cycles of amplification at 95°C for 10 s, annealing at 60°C for 20 s and extension at 60°C for 30 s. The relative expression of VWF was normalized based on the 2^−ΔΔCt method [27], with GAPDH serving as the endogenous control.

2.8 Cell counting kit 8 (CCK-8) assay

After OGD/R, BMVECs transfected with VWF-overexpressing plasmids or not were adjusted to the density of 2 × 10³ cells/well and seeded in a 96-well plate (100 μL/well). Following 24 h of incubation, the cells were subjected to DHA treatment and incubation with 10 μL CCK-8 solution (M4839, Abmole Bioscience, Houston, TX, USA) for 1 h. Thereafter, the optical density value was detected by a microplate reader (Synergy HTX, BioTek, Winooski, VT, USA).

2.9 Flow cytometry

Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (CA1020, Solarbio, China) was employed to detect cell apoptosis. In short, BMVECs treated with DHA for 24 h were collected at a concentration of 1  ×  10⁵ cells/mL, washed with cold phosphate buffered solution (PBS, abs9459, Absin, China), suspended with Binding Buffer, and centrifuged. Then, 100 μL cell suspension was incubated with 5 μL Annexin V-FITC at room temperature for 10 min away from light, and 5 μL PI for 5 min. The cells were mixed with 500 μL PBS, and apoptotic cells were analyzed using a flow cytometer (CytoFlex LX, Beckman Coulter, Miami, FL, USA).

2.10 Western blot

Total protein from rat cerebral cortex and BMVECs was extracted with a lysis buffer (BC-R327, Elabscience, China), and protein concentration was determined using BCA Protein Assay Kit (ab102536; Abcam, Cambridge, UK) according to the protocols. Equal numbers of protein samples (30 μg) were separated by 10% SDS-PAGE (P0670, Beyotime, China). After electrophoresis, the protein samples were transferred onto polyvinylidene fluoride membranes (P2438, Merck, USA), which were then treated with Tris buffered saline with Tween 20 (TBST)-dissolved in 5% skim milk (E-BC-R337, Elabscience, China) at room temperature for 1 h. Then, the membranes were cultured with diluted primary antibodies overnight (4℃). The next day, the membranes were washed with TBST and probed with secondary antibodies at room temperature for 1 h. Immunoblots were visualized by ECL Western Blotting Substrate (PE0010, Solarbio, China), and immunoreactivity was analyzed using a Tanon 5200 Imaging System (Shanghai, China). GAPDH served as the loading control. All antibodies used were as follows: VWF (1/1,000, 309 kDa, ab174290; Abcam, UK), Occludin (1/1,000, 59 kDa, ab167161; Abcam, UK), Claudin-5 (1 mg/mL, 23 kDa, AF5216; Affinity Biosciences, China), ZO-1 (1/500, 195 kDa, ab190085; Abcam, UK), LC3-I/LC3-II (1/2,000, 14, 16 kDa, ab192890; Abcam, UK), P62 (1/10,000, 62 kDa, ab109012; Abcam, UK), GAPDH (1/10,000, 36 kDa, ab181602; Abcam, UK), SIRT1 (0.125 µg/mL, 81 kDa, ab110304; Abcam, UK), FOXO1 (1/2,000, 70 kDa, ab70382; Abcam, UK), transcription factor 5 (ATF5) (1/2,000, 31 kDa, ab184923; Abcam, UK), autophagy-related gene 7 (ATG7) (1/10,000, 77 kDa, ab133528; Abcam, UK), Beclin1 (2 µg/mL, 52 kDa, ab217179; Abcam, UK), goat anti-rabbit IgG H&L (HRP) (1/2,000, ab205718; Abcam, UK), and goat anti-mouse IgG H&L (HRP) (1/2,000, ab205719; Abcam, UK).

2.11 Statistical analysis

Data were analyzed by Graphpad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) and expressed as mean ± standard deviation. Differences of CCK-8 assay results among multiple groups were compared using two-way analysis of variance (ANOVA). Differences of other experiment results were analyzed using one-way ANOVA. Values of p < 0.05 indicated significant difference.

3.1 DHA attenuated cerebral I/R injury and apoptosis in rats

As illustrated in Figure 1a, no infarction was observed in TTC-stained brain sections in the Control group. By contrast, cerebral I/R procedures induced a high infarction rate in the I/R group, but treatment with DHA in I/R-induced rats significantly reduced the infarction rate in IR + DHA group (Figure 1a, p < 0.001). The degree of rat brain injury after I/R was assessed by HE staining. The results revealed an intact tissue structure and normal neuron cells with clear membranes and nuclei in the Control group, but different degrees of interstitial edema, neuronal cell swelling, and nucleus disintegration in the I/R group. These conditions were mitigated by DHA administration in IR + DHA group (Figure 1b). In addition, TUNEL results indicated that DHA administration evidently reduced the number of TUNEL-positive cells in I/R-induced rats on day 14 following recovery (Figure 1c, p < 0.001). These results demonstrated that DHA administration exerted a protective effect by relieving the injury and apoptosis of nerve cells following IR.

Figure 1

Effects of DHA on cerebral I/R injury in vivo. SD rats were subjected to MCAO to mimic I/R injury and then administrated with DHA (0.1 mg/kg), with unprocessed rats serving as the control. (a) Brain tissues were collected from sacrificed rats to calculate infarction area by TTC staining. (b) HE staining was applied to evaluate histopathological conditions in rat brain (magnification ×100, scale bar: 100 μm). (c) TUNEL staining indicated neuron nuclei (brown). Red arrows indicated TUNEL-positive neurons after I/R injury (magnification ×100, scale bar: 100 μm). (d–j) Western blot was employed to detect relative protein levels of VWF, Occludin, Claudin-5, ZO-1, LC3I, LC3II, and P62 in rat brain tissues. GAPDH was used as the loading control. (k–n) Western blot was applied to detect relative protein levels of ATF5, ATG7, and Beclin1 in rat brain tissues. GAPDH was used as the loading control. ^** p < 0.01, ^*** p < 0.001 vs Control; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 vs I/R, ischemia/reperfusion; DHA, dihydroartemisinin; VWF, von Willebrand factor; ATF5, transcription factor 5; ATG7, autophagy-related gene 7; ZO-1, zonula occludens 1; LC3, microtubule associated protein 1 light chain 3 alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TTC, triphenyltetrazolium chloride; HE, hematoxylin and eosin.

3.2 DHA down-regulated VWF expression, protected BBB from breakdown, and inhibited autophagy in rat brain with I/R injury

Previous evidence indicated that VWF contributes to endothelial cell injury, which could be associated with autophagy-related SIRT1/FOXO1 pathway. Hence, we measured protein expressions of VWF, BBB-related proteins, and autophagy-related genes in rat brain tissues by Western blot. In the I/R group compared with the Control group, VWF, ATG7, and Beclin1 protein expressions and LC3-II/LC3-I rate were obviously elevated, yet Occludin, Claudun-5, ZO-1, P62, and ATF5 protein expressions were markedly lessened (Figure 1d–n, p < 0.01). In contrast, these trends were reversed by DHA treatment in the I/R + DHA group (Figure 1d–n, p < 0.01).

3.3 DHA promoted viability and suppressed apoptosis of BMVECs by down-regulating VWF expression

To figure out whether the effect of DHA on cerebral I/R injury is mediated by VWF, we carried out in vitro experiments using BMVECs exposed to OGD/R. The results of qRT-PCR unveiled that VWF plasmid remarkably elevated the mRNA level of VWF in BMVECs, indicating successful transfection (Figure 2a, p < 0.001). Furthermore, the results showed that OGD/R led to VWF overexpression, the effect of which was notably reversed after DHA treatment (Figure 2b, p < 0.001). In the OGD/R + DHA + VWF group compared with the OGD/R + DHA + NC group, the expression of VWF was significantly augmented (Figure 2b, p < 0.001). CCK-8 assay results unraveled that DHA treatment remarkably enhanced OGD/R-suppressed cell viability, which, however, was reversed by VWF overexpression (Figure 2c, p < 0.001). Moreover, the analysis of flow cytometry demonstrated that cell apoptosis was boosted by OGD/R, but was then inhibited after DHA treatment (Figure 2d, p < 0.001). However, VWF overexpression offset the protecting effect of DHA on cell apoptosis (Figure 2d, p < 0.001).

Figure 2

DHA promoted viability and repressed apoptosis of OGD/R-treated BMVECs by down-regulating VWF level. BMVECs isolated from newborn SD rats were transfected with VWF-overexpressing plasmids or control plasmids. OGD/R exposure was conducted on the cells with or without transfection, followed by 24 h 25 μM DHA treatment. (a and b) mRNA expression of VWF in BMVECs was determined by qRT-PCR. GAPDH served as the internal control. (c) Cell viability was assessed using CCK-8 assay. (d) Apoptotic cells were stained with Annexin V-FITC/PI and apoptosis rate was analyzed by flow cytometry. ^*** p < 0.001 vs Control; ⁺⁺⁺ p < 0.001 vs OGD/R; ^{^^^} p < 0.001 vs OGD/R + DHA + NC. BMVECs, brain microvascular endothelial cells; DHA, dihydroartemisinin; VWF, von Willebrand factor; OGD/R, oxygen–glucose deprivation/reoxygenation; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CCK-8, cell counting kit 8; NC, negative control.

3.4 DHA inhibited SIRT1/FOXO1 pathway-mediated autophagy and improved tight junction of BMVECs by down-regulating VWF expression

To explore the mechanism of DHA in tight junction of BMVECs exposed to OGD/R, we quantified protein expressions of Occludin, Claudin-5, and ZO-1 by Western blot. The results proved that prominent decreases of Occludin, Claudin-5, and ZO-1 expressions were observed in OGD/R-treated BMVECs, which was counteracted after DHA treatment (Figure 3a–d, p < 0.01). The protein levels of these three genes were down-regulated by VWF overexpression in the OGD/R- and DHA-treated BMVECs, which reversed the effect of DHA (Figure 3a–d, p < 0.01). Furthermore, to elucidate the mechanism by which DHA treatment may suppress autophagy via VWF/SIRT1/FOXO1 axis in OGD/R-treated BMVECs, we measured protein expressions of ATF5, ATG7, Beclin1, LC3-I, LC3-II, P62, SIRT1, and FOXO1 through Western blot. The results demonstrated that the protein expressions of ATF5, SIRT1, FOXO1, and P62 were decreased, and those of ATG7 and Beclin1 as well as LC3-II/LC3-I ratio were increased in the cells exposed to OGD/R, which was reversed by DHA treatment (Figure 3e–m, p < 0.01). Notably, VWF overexpression offset the effects of DHA treatment, and strikingly reduced ATF5, SIRT1, FOXO1, and P62 expressions, yet increased the protein expressions of ATG7 and Beclin1 as well as the LC3-II/LC3-I ratio in DHA- and OGD/R-treated BMVECs (Figure 3e–m, p < 0.01).

Figure 3

DHA increased tight junction proteins and inhibited autophagy in OGD/R-treated BMVECs via VWF/SIRT1/FOXO1 axis. BMVECs isolated from newborn SD rats were transfected with VWF-overexpressing plasmids or control plasmids. OGD/R exposure was conducted on the cells with or without transfection, followed by 24 h 25 μM DHA treatment. (a–d) Relative protein levels of Occludin, Claudin-5, and ZO-1 in BMVECs were measured by Western blot. GAPDH was used as the loading control. (e–i) Relative protein levels of SIRT1, FOXO1, LC3I, LC3II, and P62 in BMVECs were detected via Western blot. GAPDH was used as the loading control. (j–m) Relative protein levels of ATF5, ATG7, and Beclin1 in BMVECs were measured by Western blot. GAPDH was used as the loading control. ^** p < 0.01, ^*** p < 0.001 vs Control; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 vs OGD/R; ^{^^} p < 0.01, ^{^^^} p < 0.001 vs OGD/R + DHA + NC. ATF5, transcription factor 5; ATG7, autophagy-related gene 7; BMVECs, brain microvascular endothelial cells; DHA, dihydroartemisinin; VWF, von Willebrand factor; OGD/R, oxygen–glucose deprivation/reoxygenation; ZO-1, zonula occludens 1; LC3, microtubule associated protein 1 light chain 3 alpha; SIRT1, sirtuin1; FOXO1, forkhead box O1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control.