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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 2  |  Page : 53-63

Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats


1 Department of Physiology, Hebei Medical University; Department of Electron Microscopy Laboratory Centre, Hebei Medical University, Shijiazhuang, China
2 Department of Clinical Laboratory, Second Hospital of Hebei Medical University, Shijiazhuang, China
3 Department of Physiology, Hebei Medical University, Shijiazhuang, China
4 Department of Electron Microscopy Laboratory Centre, Hebei Medical University, Shijiazhuang, China
5 Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease, Shijiazhuang, China
6 Department of Physiology, Hebei Medical University; Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease, Shijiazhuang, China

Date of Submission22-Sep-2021
Date of Decision11-Feb-2022
Date of Acceptance18-Feb-2022
Date of Web Publication28-Apr-2022

Correspondence Address:
Yi Zhang
Department of Physiology, Hebei Medical University, Shijiazhuang 050017
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_84_21

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  Abstract 


Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of metabolic syndrome (MS) rats. This study aimed to investigate the role of adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling in CIHH effect. Six-week-old male Sprague-Dawley rats were divided into control (CON), MS model, CIHH treatment (CIHH), and MS + CIHH groups. Serum pro-inflammatory cytokines were measured. The endothelium dependent relaxation (EDR), endothelial ultrastructure and autophagosomes were observed in mesenteric arteries. The expression of phosphor (p)-AMPKα, p-mTOR, autophagy-related and endoplasmic reticulum stress-related proteins, p-endothelial nitric oxide synthase, and cathepsin D were assayed. In MS rats, pro-inflammatory cytokines were increased, EDR was attenuated, and endothelial integrity was impaired. In addition, the expression level of p-AMPKα and cathepsin D was down-regulated, but the level of p-mTOR was up-regulated. While in MS + CIHH rats, all aforementioned abnormalities were ameliorated, and the beneficial effect of CIHH was cancelled by AMPKα inhibitor. In conclusion, AMPK-mTOR signaling pathway participates in the protection of CIHH on vascular endothelium of MS rats.

Keywords: AMPK-mTOR signaling pathway, autophagy, chronic intermittent hypobaric hypoxia, endothelium dependent relaxation, metabolic syndrome


How to cite this article:
Cui F, Shi M, Hu HF, Tian YM, Zhou CM, Mi HC, Gu S, Guo Z, Zhang XJ, Zhang Y. Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats. Chin J Physiol 2022;65:53-63

How to cite this URL:
Cui F, Shi M, Hu HF, Tian YM, Zhou CM, Mi HC, Gu S, Guo Z, Zhang XJ, Zhang Y. Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats. Chin J Physiol [serial online] 2022 [cited 2022 May 21];65:53-63. Available from: https://www.cjphysiology.org/text.asp?2022/65/2/53/344269




  Introduction Top


Metabolic syndrome (MS), characterized by obesity, insulin resistance, dyslipidemia, diabetes, and hypertension, is a chronic inflammation state and a high risk of cardiovascular diseases.[1] Vascular endothelial dysfunction is an early manifestation of vascular diseases, and the pathogenic basis of cardiovascular damage in MS. Endothelium dependent relaxation (EDR) is a “gold standard evaluation” of endothelial function and a predictive hallmark of later cardiovascular diseases.[2]

Autophagy, a lysosome-related cell process, is a cell survival mechanism and plays an important role in cellular proliferation, differentiation, metabolism and stress.[3] The normal autophagy is important for the body or cell.[4] It was reported that inhibition or deficiency of autophagy function will cause an accumulation of aggregated proteins and damaged organelles,[5] exacerbates endoplasmic reticulum (ER) stress, resulting in diseases, for instance aging, cancer, neurodegeneration and heart diseases.[6],[7],[8] Autophagy contributes the endothelium function by upregulating of endothelial nitric oxide synthase (eNOS) expression, NO production, and bioavailability.[9],[10],[11] On the other hand, NO plays a controversial role in the regulation of cellular autophagy level: NO attenuates autophagy in the heart of high-salt diet rats independently of blood pressure,[12] inhibits autophagy in primary neurons or hepatic stellate cells,[13],[14] but autophagy markers are decreased in eNOS knockout mice.[15],[16]

Autophagy can be regulated through a lot of signaling pathways, in which adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathway is more important one in autophagy regulation.[17] AMPK-mTOR signaling acts as both sensor of upstream energy and activator of downstream autophagy. The phosphorylation of AMPKα may inhibit mTOR activity,[18] then triggering autophagy downstream signaling molecules in energy balance process and metabolic stress.[19] mTOR decreases eNOS activity, contributes to eNOS uncoupling and decreased NO production.[20] Inhibition of mTOR by rapamycin (RAPA) enhanced NO production,[21],[22] normalized EDR and increased the eNOS phosphorylation.[23] It is known that abnormal AMPK-mTOR signaling can lead to metabolic disorders and endothelial dysfunction associated with vascular disease in obesity and pre-diabetic/MS.[24]

The beneficial effects of chronic intermittent hypobaric hypoxia (CIHH) on body have been reported.[25],[26],[27],[28],[29] Our previous studies showed that CIHH improves endothelial dysfunction and vascular relaxation by improving autophagy flux and reducing ER stress in mesenteric arteries of MS rats.[30] Furthermore, CIHH plays a protective role in hepatic tissue by activating AMPK-mTOR signaling pathway and improving autophagy function.[31] In this study, we examined the effect of CIHH on AMPK-mTOR signaling pathways in high fat diet-induced MS rats to verify the hypothesis that AMPK-mTOR signaling pathway contributes to the protective effect of CIHH on vascular endothelium.


  Materials and Methods Top


Animals and chronic intermittent hypobaric hypoxia treatment

All experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animals, which were adopted and promulgated by the U. S. National Institutes of Health and were reviewed and approved by the Experimental Animals Welfare and Ethics Committee of Hebei Medical University.

Six-week-old male Sprague−Dawley rats (body weight 80–120 g), which provided by the Animal Center of Hebei Medical University (licence number: SYXK2020-002), were randomly divided into control (CON), CIHH treatment, MS model, and MS model plus CIHH treatment (MS + CIHH) groups. According to our previous study,[30] CON and CIHH groups were fed with chow diet (22% protein, 4% fat, and 50% carbohydrate and drinking water, specific nutrient composition per 1000 g: 99.50 g water, 216.97 g protein, 50.38 g fat, 56.87 g coarse ash, 24.00 g fiber, 13.29 g calcium, 9.17 g phosphorus), while MS and MS + CIHH groups were fed with high-fat high-fructose diet (24% protein, 12% fat, and 42% carbohydrate, specific nutrient composition: 8% lard, 2% soy flour, and 90% chow diet and 10% (wt/vol) fructose in drinking water). After induced with 16-week different diet, CIHH and MS + CIHH rats were placed in a hypobaric chamber which simulating an altitude of 5000-m (PO2 = 84 mmHg, 6 h daily for 28 days), while in the remainder of time, rats were put into a normoxic environment (18 h daily) just as those of CON and MS (24 h daily). All animals were housed in a temperature-controlled room with light/dark cycle (22°C ± 1°C) and had free access to water and food.

Enzyme linked immunosorbent assay

Blood samples were collected from the inferior vena cava of rats after fasted overnight and anesthetized by sodium pentobarbital (50 mg/kg, intraperitoneal injection). The serum was got for the measurement of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) by centrifuging at 3500 rpm for 10 min.

Ex vivo culture of arterial segments

Under sterile condition, the second- or third-order branches of mesenteric arteries were dissected and incubated in low glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 mg/mL streptomycin. After 16-h incubation with AMPKα inhibitor (Compound C, Com C, 10 μmol/L), mTOR inhibitor (RAPA, 10−6 mol/L) and lysosomal inhibitor (chloroquine, CQ, 20 μM) under the oxygenated condition (95% O2, 5% CO2) at 37°C, recording of EDR, Western blotting and transmission electron microscopy (TEM) were performed individually.

Vascular ring relaxation measurement

EDR was determined as described previously.[30] Briefly, the second- or third-order branches of mesenteric arteries were dissected, removed of the surrounding connective tissues and cut into 4–6 ring segments with length ~2.0 mm. Arterial rings were mounted on two stainless steel wires (diameter: 40 μm) in a Myograph System (Danish MyotechMyoTechnology, Aarhus, Denmark) through which the changes of isometric tension were recorded and preincubated in 5 mL of oxygenated (95% O2, 5% CO2) K-H solution at 37°C, pH 7.4, then normalized, stretched to an optimal resting tension and stabilized for 30 min. Arterial rings were exposed to 60 mM of KCl to assess their integrity of smooth muscle contractility. The functionality and integrity of vascular endothelium was valuated through 10-5 M acetylcholine (ACh)-induced vasodilation after precontracted with 10−5 M phenylephrine (PE). Vasodilation greater than 80% of maximal relaxation is considered endothelium intact and less than 10% is considered to be de-endothelialized. The accumulative concentrations of ACh (10-9 M to 10-5 M) were used to induce EDR, and concentration-response curves were built.

Vascular relaxation was expressed as a percentage reduction of maximum steady-state contraction tension induced by PE (10−5 M). The maximum ACh-induced relaxation (Emax) and the negative logarithm of ACh concentration inducing 50% Emax (pD2 values) were calculated from individual concentration-response curves by a nonlinear regression analysis.

Western blotting

Proteins from mesenteric artery tissues were extracted with a RIPA lysis/extraction buffer in the presence of protease and phosphatase inhibitor, then centrifuged at 12000 rpm (15 min, 4°C). Protein concentration was determined with a BCA kit and protein was boiled in water (15 min, 100°C). The samples (20–150 μg) were subjected to 7.5%–15% SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were incubated with 5% (w/v) nonfat milk in Tris-buffered saline for 1 h, primary antibodies overnight at 4°C (eNOS (1:200), phosphorylated (p)-eNOS (Ser1177, 1:1000), 78KD glucose-regulated protein (GRP78, 1 μg/mL), C/EBP-homologous protein (CHOP, 1:1000), Beclin-1 (1:1000), LC3B (1:1000), AMPKα (1:1000), p-AMPKα (Thr172, 1:1000), mTOR (1:1000), p-mTOR (Ser2448, 1:1000), cathepsin D (1:900), α-Tublin (1:5000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000)), and secondary antibody for 1 h at room temperature. The bands were visualized by the chemiluminescence and quantified with Image J software. The protein contents were normalized to those of GAPDH or α-Tublin on the same blot.

TEM

Mesenteric artery tissues were fixed with 4% glutaraldehyde, followed by postfixed with osmium tetroxide, dehydrated with gradient acetone, embedded in epoxy resin, and cut into ultrathin section (70 nm). After double staining with uranyl acetate and lead citrate, the sections were observed with TEM (HITACHI, HT7800).

Chemicals and solutions

PE (P6126), ACh (A6625), Com C (P5499), RAPA (V900930), CQ (C6628) and dimethyl sulfoxide (DMSO, D5879) were purchased from Sigma (St. Louis, MO, USA). Other chemicals and reagents were all of analytical grade. Com C and RAPA were dissolved in DMSO and the rest were dissolved in double-distilled water. The bath concentration of DMSO did not exceed 0.1%, which was shown to have no effects on basal tonus of the preparations or on agonist-mediated relaxation. ELISA kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China).

DMEM, FBS, penicillin, and streptomycin were purchased from Gibco (Gaithersburg, MD, USA). RIPA Lysis Buffer was obtained from BestBio (Shanghai, CN). BCA Protein Assay Kit was obtained from Tiangen Biotech (Beijing, CN). Antibodies against eNOS (ab66127) and GRP78 (ab21685) were purchased from Abcam (Cambridge, UK). Antibody against p-eNOS (Ser1177, #11156) was purchased from Signalway Antibody (Baltimore Ave, MD, USA). Antibodies against p-AMPKα (Thr172, #2535), AMPKα (#5832), p-mTOR (Ser2448, #5536), mTOR (#2983), CHOP (#2895), Beclin-1 (#3495), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against cathepsin D (NBP1-50682) was purchased from Novus Biologicals (Centennial, CO, USA). Antibody against GAPDH was purchased from Antibody Revolution (San Diego, CA, USA). Antibody against α-Tublin was purchased from Bioworld Technology (Bloomington, MN, USA). All secondary antibodies were purchased from KPL (Gaithersburg, MD, USA). Enhanced chemiluminescence kit and PVDF membranes were obtained from Millipore Corporation (Billerica, MA, USA).

K-H solution consisted of (all mmol/L) NaCl 118.0, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, glucose 11.0, pH 7.4.

Data analysis

Data are expressed as mean ± standard error of the mean. Statistical analysis was conducted using the two-way ANOVA for comparison among multiple groups of different concentrations and times, and one-way ANOVA followed by a Student-Newman-Keuls's post hoc test for comparison among multiple groups. n represents the number of animals in functional or Western blotting experiments. P < 0.05 was considered statistically significant.


  Results Top


Effect of chronic intermittent hypobaric hypoxia on the serum pro-inflammatory cytokines

Compared with CON rats, serum IL-1β, IL-6, and TNF-α were increased in MS rats (P < 0.01), but compared with MS rats, they were decreased in MS + CIHH rats [P < 0.05–0.01, [Figure 1]]. The results indicated that CIHH treatment can reduce inflammation in MS rats.
Figure 1: Effect of chronic intermittent hypobaric hypoxia on serum inflammatory cytokines in rats. (a) Serum levels of IL-1β. (b) Serum levels of IL-6. (c) Serum levels of TNF-α. Values were mean ± SEM, n = 6 in each group. *P < 0.05, **P < 0.01 versus CON; #P < 0.05, ##P < 0.01 versus MS. MS: Metabolic syndrome; CON: Control.

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Effect of chronic intermittent hypobaric hypoxia on endothelium-dependent relaxation of mesenteric arteries

Compared with CON rats (pD2: 6.27 ± 0.04; Emax: 91.33% ± 0.76%), the EDR of mesenteric artery was decreased in MS rats with concentration-relaxation curve shift rightward (pD2: 5.55 ± 0.13; Emax: 82.05% ± 2.45%, P < 0.01), and increased in CIHH rats with concentration-relaxation curve shift leftward (pD2: 6.48 ± 0.02; Emax: 96.90% ± 0.59%, P < 0.01). Compared with MS rats, the EDR in mesenteric artery was increased in MS + CIHH rats with concentration-relaxation curve shift leftward (pD2: 5.74 ± 0.49; Emax: 90.95 ± 0.76%, P < 0.01, [Figure 2]). The results indicated that CIHH treatment improved the injured EDR of mesenteric arteries in MS rats.
Figure 2: Effect of chronic intermittent hypobaric hypoxia on ACh-induced endothelium dependent relaxation in rat mesenteric arteries. (a) Original recording. (b) Concentration-response curves of ACh-induced endothelium dependent relaxation. Values were mean ± SEM, n = 5 in each group. *P < 0.05, **P < 0.01 versus CON; #P < 0.05, ##P < 0.01 versus MS. MS: Metabolic syndrome; CON: Control.

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Effect of chronic intermittent hypobaric hypoxia on expression level of adenosine mono-phosphate-activated protein kinase alpha and the mTOR proteins

Compared with CON rats, the expression level of p-AMPKα (Thr172) was decreased in MS rats (P < 0.01), and compared with MS rats, it was increased in MS + CIHH rats [P < 0.01, [Figure 3]].
Figure 3: Effect of chronic intermittent hypobaric hypoxia on the expression level of p-AMPKα (Thr172), and p-mTOR (Ser2448) in mesenteric arteries. (a) Representative blots. (b) Quantification of protein expression levels of p-AMPK. (c) Quantification of protein expression levels of p-mTOR. Values were mean ± SEM, n = 4 in each group. **P < 0.01 versus CON, ##P < 0.01 versus MS. MS: Metabolic syndrome; CON: Control; AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin.

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The expression level of p-mTOR (Ser2448) in mesenteric arteries was increased in MS rats compared with CON rats (P < 0.01) and was decreased in MS + CIHH rats compared with MS rats [P < 0.01, [Figure 3]].

The results indicated that CIHH could activate the AMPK-mTOR signaling pathway in mesenteric arteries of MS rats.

Effect of chronic intermittent hypobaric hypoxia on autophagosomes of mesenteric arteries

In CON rats, the endothelial cells of mesenteric artery were continuous and integrity. There were normal tight junctions between the endothelium, normal elastic membranes under the endothelium, and no abnormal structures in the nucleus and mitochondria [Figure 4]a. The integrity of endothelium was destroyed and uncontinuous: some completely disintegrated, others displayed expanded ER and decreased abnormal mitochondria, including fractured mitochondrial crest and the fusion of crest and membrane, in mesenteric arteries of MS rat [Figure 4]b and [Figure 4]c. The endothelial structures in mesenteric arteries of MS + CIHH rats were significantly improved, and the number of mitochondria was increased [Figure 4]d.
Figure 4: Transmission electron microscopy image. (a) Representative electron micrographs of CON rats mesenteric arteries with normal cell morphology including integrity endothelial cells, normal nucleus and mitochondria (×3.0k); (b) Disintegrating endothelial cell layer in MS rats mesenteric arteries (indicated by yellow star, ×7.0k); (c) Abnormal cell morphology including the expanding endoplasmic reticulum, fractured mitochondrial crest and the fusion of crest and membrane, were detectable in MS rats mesenteric arteries, so there were blurred structure (×7.0k); (d) Representative electron micrographs of MS + CIHH rats mesenteric arteries with integrity endothelial and normal mitochondria structures (×3.0k); (e) Image of CON rats mesenteric arteries incubated with CQ (×8.0k); (f) Image of CIHH rats mesenteric arteries incubated with CQ (×8.0k); (g) Image of MS rats mesenteric arteries incubated with CQ (×8.0k); (h) Image of MS + CIHH rats mesenteric arteries incubated with CQ (×8.0k). A partially enlarged image of mitochondria and endoplasmic reticulum was placed in the upper left corner (mitochondria were indicated by yellow arrows, endoplasmic reticulum by green arrows and tight junction by circle). Autophagosomes were indicated by red arrows. CIHH: Chronic intermittent hypobaric hypoxia; AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin; MS: Metabolic syndrome; CON: Control.

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Normally, autophagy activity is very low, and vascular endothelial cells are flat with fewer organelles, it is rare to observe autophagosomes. With the application of lysosomal inhibitor CQ to block autophagy fusion and degradation, the autophagosomes were observed. The ultrastructure of autophagosomes is the vesicle structure containing double or multilayer membranes, in which the degraded cytoplasm components are encased. The number of autophagosomes was less in MS rats compared with CON rats and was more in MS + CIHH rats compared with MS rats [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h.

Effect of chronic intermittent hypobaric hypoxia on expression of cathepsin D

The expression of cathepsin D, a lysosomes marker, was decreased in mesenteric arteries of MS rats compared with those of CON rats (P < 0.01) and was increased in those of MS + CIHH rats compared with those of MS rats [P < 0.01, [Figure 5]]. The results indicated that CIHH can activate the lysosome in mesenteric arteries of MS rats.
Figure 5: Effect of chronic intermittent hypobaric hypoxia on the expression level of cathepsin D in rat mesenteric arteries. (a) Representative blots. (b) Quantification of protein expression level of cathepsin D. Values were mean ± SEM, n = 4 in each group. **P < 0.01 versus CON, #P < 0.05 versus MS. MS: Metabolic syndrome; CON: Control.

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Influence of adenosine mono-phosphate-activated protein kinase alpha inhibitor and mTOR inhibitor on chronic intermittent hypobaric hypoxia-enhanced endothelium-dependent relaxation

EDR was decreased by AMPKα inhibitor Com C with rightward shift of concentration-relaxation curve (pD2: 5.03 ± 1.21; Emax: 39.73 ± 1.54%, P < 0.01), but increased by mTOR inhibitor RAPA with leftward shift of concentration-relaxation curve (pD2: 5.93 ± 0.12; Emax: 89.34 ± 1.33%, P < 0.01) [Figure 6] in MS + CIHH rats. The results indicated that the EDR enhancement of CIHH can be alleviated by the inhibition of AMPKα and enhanced by the inhibition of mTOR.
Figure 6: Effect of AMPKα inhibitor and mTOR inhibitor on CIHH-enhanced EDR in rat mesenteric arteries. (a) Original recording. (b) Concentration-response curves of endothelium dependent relaxation. Values were mean ± SEM, n = 5–6 in each group. *P < 0.05, **P < 0.01 versus MS; #P < 0.05, ##P < 0.01 versus MS + CIHH. CIHH: Chronic intermittent hypobaric hypoxia; AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin; MS: Metabolic syndrome; CON: Control.

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Influence of adenosine mono-phosphate-activated protein kinase alpha inhibitor and mTOR inhibitor on protein expression

AMPKα inhibitor Com C decreased the expression level of p-AMPKα (Thr172) and increased the expression level of p-mTOR (Ser2448) in mesenteric arteries of MS + CIHH rats [P < 0.01, [Figure 7]]. In addition, Com C decreased the expression level of autophagy-related Beclin-1 and LC3-Ⅱ/Ⅰ (P < 0.05–0.01), increased the expression level of ER stress-related GRP78 and CHOP (P < 0.01) and decreased the level of p-eNOS (Ser1177) in mesenteric arteries of MS + CIHH rats [P < 0.01, [Figure 8]].
Figure 7: Effect of AMPKα inhibitor and mTOR inhibitor on expression of p-AMPKα (Thr172), p-mTOR (Ser2448) in rat mesenteric arteries. (a) Representative blots. (b) Quantification of expression levels of p-AMPKα. (c) Quantification of protein expression levels of p-mTOR. Values were mean ± SEM, n = 5 in each group in measurement. *P < 0.05, **P < 0.01 versus MS; ##P < 0.01 versus MS + CIHH. AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin; MS: Metabolic syndrome; CON: Control; CIHH: Chronic intermittent hypobaric hypoxia.

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Figure 8: Effect of AMPKα inhibitor and mTOR inhibitor on expression of autophagy-related proteins, ER stress-related proteins and p-eNOS (Ser1177) in rat mesenteric arteries. (a) Representative blots. (b) Quantification of protein expression levels of Beclin-1. (c) Quantification of protein expression levels of LC3-II/I. (d) Quantification of protein expression levels of GRP78. (e) Quantification of protein expression levels of CHOP. (f) Quantification of protein expression levels of p-eNOS (Ser1177). Values were mean ± SEM, n = 4 (Beclin-1, CHOP, eNOS) and 5 (LC3-II/I, GRP78) in each group. *P < 0.05, **P < 0.01 versus MS; #P < 0.05, ##P < 0.01 versus MS + CIHH. CIHH: Chronic intermittent hypobaric hypoxia; AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin; ER stress: Endoplasmic reticulum stress; eNOS: Endothelial nitric oxide synthase; MS: Metabolic syndrome; CON: Control.

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On the contrary, mTOR inhibitor RAPA increased the expression level of p-AMPKα (Thr172) and decreased the expression level of p-mTOR (Ser2448) in mesenteric arteries of MS + CIHH rats [P < 0.01, [Figure 7]]. In addition, RAPA increased the expression level of autophagy-related Beclin-1 and LC3-Ⅱ/Ⅰ (P < 0.01), decreased the expression level of ER stress-related GRP78 and CHOP (P < 0.05–0.01), and increased the level of p-eNOS (Ser1177) in mesenteric arteries of MS + CIHH rats [P < 0.01, [Figure 8]].

The results indicated that autophagy-related proteins can be regulated by the AMPK positively and mTOR negatively, and the effect of CIHH on proteins expression can be decreased by AMPKα inhibitor Com C but increased by mTOR inhibitor RAPA.


  Discussion Top


In this study, the effect of CIHH on vascular endothelium function, autophagy, and signaling molecules was investigated in high-fat-high-fructose induced MS rats. The result showed that the inflammation was enhanced, EDR was attenuated, endothelial integrity was impaired, the level of p-AMPKα was down-regulated, while the level of p-mTOR was up-regulated in MS rats. Importantly, the aforementioned deviations in MS rats were reverted or improved by CIHH treatment, which was cancelled by an AMPKα inhibitor and enhanced by mTOR inhibitor. It suggests that the beneficial effect of CIHH on vascular endothelium and autophagy is related with AMPK and mTOR molecules.

Autophagy flux, including formation and degradation of autophagosomes, reflects autophagy function for its dynamic process of formation and degradation of autophagosomes more precisely.[6],[7],[8] The inhibition or deficiency in autophagy flux results in an accumulation of aggregated proteins and damaged organelles and exacerbation of ER stress and diseases. It was known that high fat diet affects the formation of autophagosomes and autophagy flux. In chronic or acute hyperlipidemia the fusion capacity of autophagosome and lysosome was decreased, called as blocked autophagy flux, leading to autophagy dysfunction.[32] Many therapeutic agents such as curcumin, spermidine, and trehalose, prevent or reverse age-related arterial dysfunction and cardiovascular disease through enhancing autophagy flux.[33],[34],[35] The autophagy flux can be evaluated by using lysosome inhibitors to block autophagy fusion and degradation in the lysosome to observe the changes of autophagosome and autophagy-related proteins: the large change of autophagosome and autophagy-related proteins indicates the obstacle in autophagy induction, and the small or no change indicates the obstacle in autophage degration.[5] Our previous study showed that the autophagy function was decreased with an increasing autophagy-related protein expression in mesenteric artery of MS rats which due to obstruction of autophagic degradation process, and CIHH improved the autophagy function with a decreases of autophagy related proteins expression through enhancing autophagic degradation.[30] In this study, the decreased expression of cathepsin D, lysosomes marker, was reversed by CIHH treatment in MS rats, which implied the reduced degradation function; combined with the smaller change of autophagosome in MS, we can further confirmed that the increased expression of autophagy-related protein was owing to a reduction of autophagic degradation at later stages, not to the activation of autophagy function in MS rats.

AMPK is composed of three subunits, in which the phosphorylation of Thr172 site of the alpha subunit leads to the activation of kinase. AMPK can inhibit mTOR activity through phosphorylating the Thr2446 site of mTOR directly or inhibiting the phosphorylation of Ser2448 site of mTOR indirectly.[18] AMPK-mTOR signaling pathway is one of upstream molecular of important regulatory autophagy flux, mainly through inhibiting mTOR activity[18] and triggering autophagy downstream signaling molecules,[19],[36] which plays an important role in glucose transport, lipid metabolism, inflammation, ER stress and vascular function.[17] The link between autophagy or AMPK-mTOR and vascular functions has been described in both in vitro and in vivo studies. It was reported that AMPK activity, NO synthesis rate, and EDR was reduced in genetical obese animals, such as OLETF rats[37] and obese Zucker rats.[38] In endothelium of high fat diet-induced obese mice and rats, AMPK-mTOR pathway was changed abnormally, along with the reduced eNOS phosphorylation and NO availability,[39] and metabolic disorders and endothelial dysfunction.[24] In defect of endothelial autophagy induced by low shear stress, AMPKα isinhibited and mTOR is activated with a blockade of the autophagic flux.[10] In this study, the abnormal changes of EDR, p-AMPK and p-mTOR proteins were reversed by CIHH treatment in MS rats, implying that CIHH protects against vascular endothelial injury through AMPK-mTOR signaling pathway. So as to clarify the relationship between AMPK-mTOR signaling and CIHH effect, AMPKα inhibitor Com C and mTOR inhibitor RAPA were used, respectively. The results showed that the expression level of p-mTOR protein was increased, and the expression level of autophagy-related proteins was decreased by Com C. Meanwhile, the expression level of ER stress-related proteins, p-eNOS, and EDR were decreased in mesenteric arteries of CIHH-treated MS rats. Otherwise, the opposite effect was produced by RAPA. So, the important role of AMPK-mTOR signaling pathway in the CIHH effect was further confirmed.

A great number of studies have demonstrated the close relationship among autophagy, inflammation and AMPK: pro-inflammatory cytokines can reduce autophagy through p38α MAPK signaling, and autophagy can suppress proinflammatory process through the regulation of innate immune signaling and inflammasome activity.[40],[41] For example, the deficiency of autophagy can increase IL-1β and IL-18 production associated with intestinal inflammation and inflammasome hyperactive in ATG16L-deficient mouse model.[42] Also, deficiency of autophagy can accelerate atherogenesis in hypercholesterolemic ApoE−/− mice, which is related to an inflammatory and defects in endothelial alignment.[7] In addition, age-associated reduction of autophagy can increase ER stress and pro-inflammatory cytokine production in old adipose tissue.[43] The autophagy activation under laminar shear stress can protect endothelial cells against oxidative stress, upregulate endothelial NO synthase expression, and inhibit endothelial inflammation.[10] Furthermore, the research has demonstrated that AMPK-activating drugs produce anti-inflammatory effects[44] through AMPK signaling in skeletal muscle.[45] The present study showed that the level of pro-inflammatory cytokines increased in MS rats but decreased in CIHH-treated MS rats, implying that CIHH may antagonize inflammation by activating AMPK-mTOR signaling and enhancing autophagy in MS rats.


  Conclusion Top


Based on our previous result that CIHH protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of MS rats, this study further demonstrated that CIHH protects vascular endothelium through activating AMPK-mTOR signaling pathway, then ameliorating autophagy and antagonizing inflammation in mesenteric arteries of MS rats [Figure 9]. This study provide direct evidence for further molecular or genetic study of CIHH protection on vascular endothelium.
Figure 9: Schematic summarizing possible protective mechanisms of CIHH on vascular endothelium. CIHH could activate AMPK, inhibit mTOR, which leads to amelioration of autophagy and ER stress, then increase eNOS activity, and improve EDR in MS rats. Red arrow: Activation; green arrow: Inhibition. CIHH: Chronic intermittent hypobaric hypoxia; AMPK: Adenosine mono-phosphate-activated protein kinase; mTOR: Mammalian target of rapamycin; ER stress: Endoplasmic reticulum stress; eNOS: Endothelial nitric oxide synthase.

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Authors' contributions

F. C, M. S and H. F. H performed the experiments and drafted the manuscript; Y. M. T took care of animals; C. M. Z prepared the images; H. C. M performed ELISA experiment; S. G, Z. G, and X. J. Z analyzed the data; Y. Z was responsible for conception and design of the research, revised the final manuscript. All authors contributed to the article and approved the submitted version.

Financial support and sponsorship

This work was financially supported by the National Basic Research Development Program of China [grant number: 2012CB518200], the National Natural Science Foundation of China [grant numbers: 31671184, 31271223, 31071002], Hebei Province Higher Education Science and Technology Research Project [grant number: QN2019014], and the Natural Science Foundation of Hebei Province [grant number: C2020206050].

Conflicts of interest

There are no conflicts of interest.



 
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