17β estradiol activates autophagy and attenuates homocysteine mediated inflammation in endothelial cells through PI3K AKT MTOR signaling

Abstract

The accumulation of plasma Hcy has been linked to vascular inflammation, which can result in vascular dysfunction. Autophagy has been the subject of extensive research in the context of hypertension-related vascular inflammation injury. As demonstrated in a preceding study, the protective effect of estradiol against vascular injury is attributable to its impact on autophagy. The present study has been designed to elucidate the epidemiological relationships between estrogen, homocysteine (Hcy), high-sensitivity C-reactive protein (hs-CRP), and their synergistic effects on the development of hypertension at the population level. In addition, the study will mechanistically investigate how estrogen-mediated autophagy counteracts Hcy-induced inflammatory injury in human umbilical vein endothelial cells through experimental validation. A case-control study was conducted on 559 female patients at Hunan Provincial People’s Hospital, who were divided into hypertensive and normotensive groups. The investigation revealed that the age, Hcy and Hs-CRP concentration of the hypertensive group exceeded those of the normotensive group (P < 0.001). Conversely, the estrogen level exhibited the opposite trend (P < 0.001). The logistic regression analysis indicated that plasma estrogen levels are a beneficial factor in relation to hypertension. Conversely, plasma Hcy and Hs-CRP levels have been shown to be detrimental, and there is an interaction between the two. In the preliminary experiment, the presence of 17β-estradiol (E2) was observed to stimulate the proliferation of Hcy-treated umbilical vein endothelial cells, whilst concomitantly exerting an inhibitory effect on inflammation. The present study demonstrated that Hcy inhibited autophagy in cultured human umbilical vein endothelial cells (HUVECs), while E2β reversed this inhibition. In conclusion, the present study demonstrated that 17β-estradiol attenuates homocysteine-induced inflammatory injury in HUVECs through the activation of autophagy via the PI3K/Akt/mTOR signalling axis.

Introduction

Hypertension has been designated a global epidemic and chronic disease, and it is recognized as the basis of various cardiovascular diseases. The sustained elevation of blood pressure is associated with a series of pathophysiological alterations to the vascular endothelium. The vascular endothelium is not a static barrier to blood cells, pathogens and chemical signals. Instead, it has been demonstrated that the vascular endothelium actively converts the circulating mediator into a tissue response and rapidly changes to physiological stress¹. The homocysteine (Hcy) has been demonstrated to be a causative agent in the accumulation of this substance in the blood, a phenomenon that has been observed to occur as a consequence of either genetic or nutritional deficiencies. This may result in hyperhomocysteinemia (HHcy), which has been demonstrated to induce endothelial dysfunction, a hallmark of elevated blood pressure and atherosclerosis³. However, the precise mechanism remains to be elucidated; it is hypothesized to be associated with autophagy⁴.

Autophagy is a highly conserved process that maintains homeostasis by lysosomal degradation of damaged organelles and proteins under stress⁶. It plays an important role in endothelial cell homeostasis, vascular remodelling and cardiovascular disease⁷. Autophagy has recently been the focus of research in the context of vascular disease, in which it functions as an adaptive protective effect against apoptosis⁹. Nevertheless, it is conceivable that autophagy may be excessively activated, thus resulting in the death of endothelial cells. This, in turn, may lead to inflammation and thrombosis.

It is well established that premenopausal women exhibit relative cardiovascular protection, signifying that the risk of cardiovascular disease is considerably lower in women compared to men of the same age. This gender discrepancy has given rise to a contentious debate, and it is evident that this phenomenon is closely associated with the beneficial effects of sex hormones, particularly estrogen¹⁰. The mechanism of action of estrogen may involve inhibiting inflammation and oxidative stress, and affecting apoptosis¹². For instance, estradiol-17β has been observed to modulate autophagy through the p53 pathway, thereby impeding the senescence of human umbilical vein endothelial cells¹⁵. However, the effects of estrogen on autophagy remain to be fully elucidated under various stress conditions, particularly in the context of Hcy treatment.

Our research is divided into two parts: (1) to explore the effects of estrogen, Hcy, and high-sensitivity C-reactive protein (Hs-CRP) and their interactions on hypertension in a clinical epidemiological study; (2) to explore the protective effect of 17β-estradiol against Hcy-induced inflammation in HUVECs and its molecular mechanism.

Materials and methods

Study population

The subjects were all recruited from the Hunan Provincial People’s Hospital. A case-control design was employed, enrolling female participants aged 30–90 years, comprising patients with hypertension (cases) and individuals with normal blood pressure (controls). Participants were excluded if they had taken antihypertensive medication, had a history of malignant tumours, kidney disease, or endocrine disorders. Ultimately, 559 eligible participants were included in the study sample. Data on demographic characteristics and potential risk factors were collected. The research was approved by the Ethics Committee and all participants provided written informed consent.

Data collection and disease definition

The self-made questionnaire primarily covered demographic characteristics, lifestyle, past medical history, family medical history, and physical examination findings. All selected patients with hypertension and HHcy were diagnosed by qualified clinical physicians. Blood pressure was measured using a conventional mercury sphygmomanometer. Adults with a systolic blood pressure (SBP) ≥ 140 mmHg and/or a diastolic blood pressure (DBP) ≥ 90 mmHg, or those with a history of antihypertensive medication use, were defined as hypertensive patients. Plasma homocysteine (Hcy) concentration ≥ 15 µmol/L was defined as Hyperhomocysteinemia (HHcy).

Blood samples and laboratory testing

Participants fasted for 8–12 h prior to blood sample collection. Venous blood samples were collected in the morning after an overnight fast using anticoagulant-treated tubes. The samples were transported to the laboratory under refrigeration (4 °C) for analysis. Total plasma homocysteine (Hcy) concentration was measured using the enzymatic cycling assay method (Meikang Hcy Detection Kit). Additional biochemical indicators—including plasma estradiol and high-sensitivity C-reactive protein (Hs-CRP)—were analyzed with a fully automated biochemical analyzer (Roche Cobas c8000).

Drugs

17β-estradiol (E2β), Homocysteine(Hcy) were purchased from sigema. MK2206(an autophagy inhibitor), 3-MA (a protein kinase B (Akt) inhibitor) were purchased from selleck.

Assessment of HUVECs viability by cell counts

Cell proliferation was assessed by direct cell counting. HUVECs were seeded in 6-well plates at a density of 1 × 10⁵ cells/well and cultured overnight. At designated time points after E2β and Hcy treatment, cells were trypsinized to generate single-cell suspensions. Trypan blue (final concentration 0.04%) was added, and viable cells were counted manually using a hemocytometer under a phase-contrast microscope.

HUVECs culture and treatment

The umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. For experiments, cells were pretreated with 17β-estradiol (100, 200, 400, or 800 nM) in medium containing 5% FBS for 12 h. After removal of the pretreatment medium, cells were exposed to fresh medium with 5% FBS containing both 17β-estradiol (maintained at pretreatment concentrations) and homocysteine (2, 4, or 8 µM) for an additional 36 h. Cell analyses were subsequently performed as described below.

Assessment of HUVECs viability

Cell proliferation was assessed using a CCK-8 assay. Cells were seeded in 96-well plates at 5,000 cells/well and allowed to adhere overnight. Following Hcy and estrogen treatment, 10 µl of CCK-8 reagent was added to each well. After incubating at 37 °C for 2 h in a humidified 5% CO₂ atmosphere, absorbance was measured at 450 nm using a microplate reader with a reference wavelength of 650 nm.

Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) is used to detect autophagic vesicles in our research and is the most reliable method for autophagy detection¹⁶. HUVECs in logarithmic growth phase were pretreated with 4 µM homocysteine (Hcy) for 1 h, followed by co-treatment with 200 nM 17β-estradiol (E2) for 48 h in 6-well plates. The cells were collected and fixed with electron microscope solution. The 50 nm ultra-thin section was cut with an ultra-thin microtome and was observed with a Philips CM1220 electron microscope under 80kv voltage.

LDH and TNF-αLeakage rate

Cell damage was determined by the amount of LDH released from the cell culture medium. The LDH activity in the medium was measured using a LDH activity assay kit according to the manufacturer’s instructions. Inflammation was determined by the concentration of released TNF-α in HUVECs culture medium. The TNF-α concentration in the culture medium was measured using a TNF-αassay kit according to the manufacturer’s instructions.

Western blot analysis

The total protein was extracted using RIPA¹⁷. Equal amounts of protein (40 µg) from each group were separated via SDS-PAGE, and electrophoresis was transferred to PVDF membrane. After blocking with 5% skim milk for 1 h, the PVDF membrane was treated with the primary antibodies GAPDH (abcam, UK, 1:1000 dilution), AKT and P-AKT, mTOR and P-mTOR, LC3I and LC3II. Then we washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) for 1 h at room temperature, and observed via enhanced chemiluminescence. Protein levels were determined using ImageQuant LAS 500 (General Electric Company, America) and analyzed using Image J software.

Statistical analysis

All analyses were performed using SPSS 22.0. Continuous variables with normal distribution are presented as mean ± standard deviation (SD) and compared by t-tests between two groups. Categorical variables are expressed as frequencies (%) and analyzed by χ² tests. Binary logistic regression was used to assess the association between plasma estrogen concentration and hypertension, with odds ratios (ORs) and 95% confidence intervals (CIs) reported. Statistical significance was defined as two-sided P < 0.05.

The basic experiment section was repeated at least three times. Experimental data were presented as mean ± standard error of the mean (SEM), and experimental data were statistically analyzed using SPSS 22.0. Data from multiple groups were compared using one-way analysis of variance (ANOVA) and the Bonferroni test was used to compare differences between groups. All statistical analysis P<0.05 considered the difference to be statistically significant.

Method declaration

All methods were carried out in accordance with the relevant guidelines and regulations.

Results

Characteristics of study subjects

A total of 559 participants have provided complete demographic, blood pressure and plasma biochemical data. The demographic profile of the participants revealed that the majority were above the age of 55 (74.6%), many were in a state of menopause (87.3%), and a significant proportion were diagnosed with hypertension (80.7%).(Table 1).

Table 1. Baseline data of participants in hunan, 2018–2019.

Variables	Groups	N (%)
Age	<55y	142(25.4)
	≥ 55y	417(74.6)
Menopause	Yes	488(87.3)
	No	71(12.7)
HHcy	Yes	361(64.6)
	No	198(35.4)
Hypertension	Yes	451(80.7)
	No	108(19.3)
Total		559

HHcy: Hyperhomocysteinemia;.

All participants were divided into two groups which are hypertension group and the normotensive group. The age, plasma Hcy and plasma Hs-CRP concentration of hypertensive population are significantly higher than normotensive population. However, plasma estrogen concentration is lower in hypertensive patients than normotensive population (all P < 0.05). (Table 2)

Table 2. Comparison of baseline data of female participants with hypertension and non-hypertensive population.

Variables	Normotensive	Hypertension	p
Age(y)	52.05 ± 14.57	65.61 ± 10.85	0.0001
Hcy(µmol/L)	10.42 ± 3.21	14.50 ± 4.78	0.0001
Estradiol(pg/ml)	38.92 ± 26.05	16.14 ± 17.40	0.0001
Hs-CRP(mg/L)	0.18 ± 0.23	0.28 ± 0.31	0.002

Comparison among 2 groups was performed by a T test. Comparison among 2 groups was performed by a Chi square test.

HBP= Hypertension; Hhcy = Hyperhomocysteinemia; HS-CRP= hypersensitive C-reactive protein.

Plasma estrogen and Hs-CRP levels in different Hcy and blood pressure groups

The participants were divided into four groups according to whether they had hypertension with HHcy. The plasma estrogen concentration in the hypertension and HHcy group was the lowest (10 ± 11.95) pg/ml. Furthermore, the highest level of estrogen was observed in the non-hypertensive group with N-Hcy, which was (40 ± 19.20) pg/ml, representing a fourfold increase compared to the lowest group. And the difference between the two groups was statistically significant. (P < 0.05) (Fig. 1A).

Fig. 1 [Images not available. See PDF.]

Plasma estrogen and Hs-CRP levels of different HCY and hypertension groups. The participants were divided into four groups according to whether they had hypertension and hyperhomocysteinemia (HHcy). Plasma estrogen concentration in different groups (A). Plasma Hs-CRP concentration in different groups (B). HHcy: hyperhomocysteinemia.

Same as above, the participants were to be divided into the same four groups. The participants in the study demonstrated the highest levels of Hs-CRP (3.71 ± 9.42 mg/L) in the group that exhibited hypertension and HHcy. Participants in the non-hypertensive group with high homocysteine had the lowest levels of Hs-CRP, with a mean value of 1.41 ± 0.81 mg/L. This was one-third of the value observed in the group with the highest levels of HHcy.And the difference between the two groups was statistically significant (P < 0.05). (Fig. 1B)

The effect of the interaction of estrogen and other factors on hypertension events

The dependent variable was hypertension status. Plasma estrogen, Hcy and Hs-CRP were independent variables for logistic regression analysis. Because hypertensive events and plasma estrogen, Hcy, Hs-CRP concentration all are affected by age¹⁸. Adjusted age levels, the results showed that estrogen is a protective factor for hypertension events and plasma Hcy and Hs-CRP are risk factors for hypertension. (Table 3)

Table 3. Multivariate logistic regression analysis of the relationship between estrogen, hcy, Hs-CRP and hypertension.

	OR	CI	P
Estradiol (pg/uL)	0.974	0.961–0.988	0.0001
Hcy(µmol/L)	1.282	1.175–1.399	0.0001
Hs-CRP(mg/L)	5.354	1.757–16.313	0.003

Adjust age levels.CI=confidence interval; OR=odds ratio.

Hcy= Homocysteine; HS-CRP= hypersensitive C-reactive protein.

As we all know, plasma Hcy and Hs-CRP are recognized harmful substances in hypertension and vascular endothelium²². To investigate estrogen’s protective mechanisms, we analyzed its interactions with these biomarkers. The results showed that estrogen demonstrates a positive interaction with both homocysteine and high-sensitivity C-reactive protein. However, estrogen levels were negatively correlated with Hcy and also exhibited a negative correlation with Hs-CRP (Table 4). In other words, in addition to protecting blood vessels alone, estrogen can also reduce the occurrence and development of hypertensive events by regulating the plasma Hcy and Hs-CRP concentration.

Table 4. The interaction between Estrogen and hcy, Hs-CRP affects hypertension.

	OR	CI	P
Hcy(µmol/L)	0.997	0.996–0.998	0.0001
Hs-CRP(mg/L)	0.791	0.713–0.878	0.0001
Hcy(µmol/L)* Hs-CRP(mg/L)	1.024	1.014–1.034	0.0001

Age and education levels were adjusted for.

CI=confidence interval, OR=odds ratio.

Hcy= Homocysteine; Hs-CRP= hypersensitive C-reactive protein.

Estrogen reduces Hcy-induced damage in HUVECs

Chronic inflammation of blood vessels is the basic pathological change of hypertensive and atherosclerosis²⁴. The results of our population-based research also initially hinted at this point. In order to explore whether estrogen protects the vascular endothelium by affecting vascular inflammation, we further designed basic experiments at the cellular level.

The growth status in HUVECs was detected by Cell proliferation activity, LDH release, and TNF-α secretion. As shown in Fig. 2A, the Hcy treatment of cells reduced cell proliferation activity, and it was concentration-dependent. In addition, E2 treatment reversed the effect of Hcy on proliferation. When the concentration of E2 treatment was 200 nM, the protection was the greatest. And the following experiments were treated with this concentration (Fig. 2B). In addition, E2 treatment also reversed the harmful effects of Hcy-induced damage and inflammation (Fig. 2C, D). It also indicated that estrogen reduces Hcy-induced inflammation.

Fig. 2 [Images not available. See PDF.]

Estrogen protects against Hcy induced injury in HUVECs. HUVECs were treated with 0, 2, 4, 8µM Hcy (A) and with 0, 100, 200, 400 ang 800 nM 17-β estradiol (E2) (B). CCK8 detection kit was used to detect cell proliferation activity. (C) The leakage rate of LDH in the cell culture supernatant was measured with the LDH detection kit. (D) The leakage rate of TNF-α in the cell culture supernatant was measured with the TNF-α detection kit. Values are the means ± SEM from 3 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. Hcy. E2β: 17-β Estradiol; Hcy: Homocysteine.

Estrogen induces autophagy under Hcy-treated in HUVECs

The TEM was used to evaluate the autophagy and confirmed the effects of E2 on autophagy in HUVECs (Fig. 3). Hcy treatment caused cell damage and inflammation but did not affect autophagy in HUVECs (Fig. 3E, F). Adding E2 on this basis increased autophagosomes and activated autophagy in HUVECs. (Fig. 3G, H).

Fig. 3 [Images not available. See PDF.]

Estrogen induces autophagy in HUVECs. Transmission electron microscopy was used to evaluate autophagy induced by 17-β Estradiol Magnification, 24,500×. Scale bar, 2 μm. The black arrows represent autolysosomes (A-E).

Estrogen inhibites the PI3K-AKT-MTOR-LC3 signaling pathway in HUVECs

To further study the mechanisms by which E2 protects HUVECs against damage caused by Hcy, western blot was used to examined possible signal pathways. In HUVECs, Hcy significantly increased the protein expression levels of p-mTOR and p-AKT, while E2 treatment further reduced these expression levels (Fig. 4A, B,C, D). Meanwhile, in order to determine the association between E2 and autophagy in HUVECs, we detected the LC3II/I ratio and P62 expression levels in the cells, all of which are indicators of autophagy. Hcy treatment elevated the expression level of the autophagy marker p62 and decreased the protein expression level of LC3II/I, which was reversed by E2 treatment (Fig. 4E, F,G, H).

Fig. 4 [Images not available. See PDF.]

Estrogen inhibites PI3K-Akt-mTOR pathway in HUVECs. Western blot and quantitative analysis AKT, mTOR, P62, LC3 expression in HUVECs (A-E). Expression of LC3 І/II protein in HUVECs stimulated by E2β at different concentration (F). Values are the means ± SEM from 3 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. Hcy. E2β: 17-β Estradiol; Hcy: Homocysteine. mTOR: mammalian target of rapamyein, Akt: protein kinase B.

E2 induces autophagy by regulating PI3K-Akt-mTOR

In order to determine the pathways related to autophagy in the above process, HUVECs were treated with upstream AKT inhibitor MK-2206 and upstream autophagy inhibitor 3-MA. After Pretreatment with 3-MA for 1 h, the results showed that autophagy was inhibited and the expression of p-AKT, P-mTOR, LC3II/I and p62 protein by E2-treatment was reversed in Hcy treated-HUVECs (Fig. 5). Following a 0.5-hour pre-treatment with MK-2206, inhibition of AKT phosphorylation caused a certain recovery of the effect of Hcy on autophagy in HUVECs (Fig. 5). These results indicate that E2 inhibits the PI3K-AKT-mTOR-LC3 pathway in Hcy-treated HUVECs.

Fig. 5 [Images not available. See PDF.]

PI3K-Akt-mTOR pathway is involved in E2β-mediated anti-HUVECs inflammation. MK2206 is protein kinase B(AKT) inhibitor. 3-MA is 3-Methyladenine, PI3K related autophagy inhibitors.Western blot and quantitative analysis of HUVECs AKT, mTOR, P62, LC3 expression (A-D). Elisa kit and quantitative analysis of LDH and TNF-α leakage rate in HUVECs (E, F). CCK8 detection kit was used to detect cell proliferation activity (G). Values are the means ± SEM from 3 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. Hcy. mTOR: mammalian target of rapamyein, Akt: protein kinase B, MK2206: protein kinase B inhibitor.

E2 resists Hcy-induced damage and inflammation by regulating the PI3K-Akt-mTOR pathway

To further explore whether blocking PI3K-Akt-mTOR change the protective of E2 in Hcy-damaged HUVECs, we tested proliferation, injury and inflammation in HUVECs. The proliferative activity, injury and inflammatory factor levels of E2 treatment were also reversed by 3-MA pretreatment in HUVECs (Fig. 6). In addition, the proliferative activity, injury and inflammatory factor levels after Hcy treatment were also reversed by MK-2206 pretreatment in HUVECs (Fig. 6A-C). The results suggested that in the stress environment of Hcy treatment, estrogen promoted autophagy to play a protective role through PI3K-Akt-mTOR pathway in HUVECs. It has also been determined that Hcy-induced inflammatory damage of HUVECs was through Akt-mTOR-LC3 pathway.

Fig. 6 [Images not available. See PDF.]

Cells were treated with MK2206 or 3-MA with or without E2β or/and Hcy to assess the role of PI3K-AKT-mTOR pathway in E2β stimulation of HUVECs cell proliferation (A), damage (B) and inflammation (C). Values are the means ± SEM from 3 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. Hcy.

Discussion

Our population survey revealed that plasma estrogen is a protective factor for hypertension and there are negative interactions with plasma Hcy and Hs-CRP respectively. Further mechanism studies clarified that E2 exposure reduces Hcy-induced damage and inflammation in HUVECs, which promotes autophagy through the pi3k-akt-mtor signaling pathway. This may provide new insights into the mechanism of estrogen replacement therapy.

Menopausal events are widely believed to be related to increased risk of cardiovascular disease²⁶. However, the risk and benefit of menopause hormone therapy (MHT) for cardiovascular disease has always been a hot topic of controversy among scholars²⁷. Menopause management and menopausal hormone treatment guidelines in China point out that starting MHT as soon as possible after estrogen deficiency can protect women’s cardiovascular⁵. However, the specific mechanism of estrogen is still unclear and there is an urgent need to clarify the primary prevention mechanism of MHT for menopausal cardiovascular disease. According to the clinical, our research explored the effect and mechanism of estradiol in intervention of hypertension. The occurrence and development of hypertension are inseparable from long-term inflammation infiltrating blood vessels. Our study has the same conclusion, data analysis shows that high estradiol is a protective factor and the inflammatory molecule Hs-CRP is a harmful factor for hypertension events. There is an interaction between estrogen and Hs-CRP. In summary, estrogen may play a vascular protective effect by inhibiting the release of Hs-CRP.

Atherosclerosis (AS) is a chronic vascular inflammatory disease, which is an abnormal response of the vascular wall to various injuries, and has the characteristics of classic inflammatory degeneration, exudation and hyperplasia²⁹. The inflammatory changes of vascular endothelial cells are the initial stage and inflammatory factors accelerate the pathological process³⁰. It is currently believed that plasma Hcy is the pathophysiological basis of vascular remodeling³¹. Our study showed that Hs-CRP concentration in patients with HHcy and hypertension increased significantly, while estradiol concentration decreased (Fig. 1). Zhang et al. also found that estradiol can inhibit the release of inflammatory factors in LPS-induced Raw 264.7 cells by via activating PI3-K/Akt signal. Autophagy plays an important role in vascular inflammation¹⁴. For this reason, our study also verified this point, which estradiol promotes autophagy and thus inhibits Hcy-induced inflammation in HUVECs.

Autophagy is a self-digestion process mediated by intracellular lysosomes, which plays an important role in cancer, neurodegenerative diseases and cardiovascular diseases³³. The scaffold protein p62/SQSTM1 serves as a selective autophagy receptor that recognizes polyubiquitinated cargo through its ubiquitin-associated (UBA) domain while simultaneously binding LC3-family proteins via its LC3-interacting region (LIR). During autophagosome maturation, p62 facilitates cargo sequestration by tethering ubiquitinated substrates to nascent autophagosomal membranes. Following autophagosome-lysosome fusion, the p62-cargo complex undergoes degradation by lysosomal hydrolases. Consequently, p62 protein abundance inversely correlates with autophagic flux, making it a validated biomarker for monitoring autophagy progression through quantitative Western blot analysis. LC3, a pivotal regulator of autophagy, is universally recognized as a gold-standard biomarker for assessing autophagic activity. This protein exists in two distinct isoforms: cytosolic LC3-I and membrane-bound LC3-II. During autophagy initiation, LC3-I undergoes a ubiquitin-like modification process mediated by the ATG conjugation system (ATG4, ATG7, and ATG3), where it becomes covalently linked to phosphatidylethanolamine (PE) through a process termed lipidation. This post-translational modification converts LC3-I to its lipidated form LC3-II, which subsequently becomes stably integrated into elongating autophagosomal membranes³³. The LC3-II/LC3-I ratio has been extensively validated as a quantitative indicator of autophagosome formation dynamics, as demonstrated in numerous studies.Estrogen has been reported to partly regulate various activities of the reproductive organs by regulating autophagy³⁵.The autophagy activity of uterine epithelial cells in ovariectomized rats and perimenopausal rats was higher than normal. Studies have shown that E2 is a protective factor in lipopolysaccharide (LPS)-induced endotoxemia in castrated rats by promoting autophagy³⁷. However, another report showed that E2 can protect cardiomyocytes from LPS-induced damage by inhibiting autophagy¹². These studies indicate that estrogen-mediated autophagy has opposite effects on LPS-induced damage under different conditions. Under the treatment of starvation or cytotoxic substances, estrogen promotes cell survival by promoting autophagy¹². In general, the effect of estrogen on autophagy varies with different conditions. Estrogen can promote survival or death in different tissues of different stressors through autophagy, which may be related to the different expression and distribution of estrogen targets. Our research confirmed that estrogen plays a protective effect in Hcy-damaged HUVECs by promoting autophagy.

According to reports, autophagy induced inflammation through a variety of pathways. For example, resveratrol induced autophagy through cAMP signaling pathway and inhibited endothelial cell inflammation³⁸. Kaempferol up-regulated autophagy and reduced ox-LDL-induced apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway in HUVECs³⁸. MicroRNA-126 and microRNA-155 restore autophagic flux and reduced endothelial cell damage by inhibiting PI3K/Akt/mTOR³⁹. Therefore, autophagy may be important targets for the prevention and treatment of vascular endothelial injury and the PI3K/Akt signaling pathway is an important negative regulatory pathway for autophagy⁴⁰. In general, estrogen regulates the occurrence of autophagy is related to the state of the cell. Estrogen promotes survival or death in different tissues of different stressors through autophagy. Our results also confirmed once again that estradiol promotes autophagy through the PI3K/AKT signaling pathway and inhibits inflammatory in Hcy-damaged HUVECs.

Shortly, estrogen participates in the prevention and treatment of cardiovascular diseases by anti-atherosclerosis, regulating vascular function, protecting endothelium. However, the deficiency of clinical diagnosis estrogen cannot be ignored. Our research once again confirmed that estradiol is negatively correlated with hypertension and Hs-CRP is positively correlated with it. The mechanism may be that estradiol promotes autophagy through the PI3K-AKT signaling pathway in Hcy-stressed HUVECs, which affects the process of vascular endothelial disease. However, due to limited conditions, experiments cannot completely replace the physiological and pathological environment at the biological level, so it is still unknown whether estradiol passes through other channels. To this end, we will further conduct animal experiments to provide reference data for the use of MHT in the primary prevention of menopausal cardiovascular disease.

Limitations and future studies

We realized the limitation of this study regarding the autophagy analysis. Cell autophagy was examined only by the method of Western blot, other methods, such as using electron microscopy, are planned in our future studies. And other conventional autophagy factors will be added for further investigation in future experimental studies.

Conclusion

Our research confirmed that there is an interaction between human plasma estrogen, Hcy and Hs-CRP, which affects the occurrence of hypertension. And it was discovered that E2 induces autophagy and alleviates inflammation through the PI3K-AKT-mTOR pathway to reduce Hcy-induced umbilical vein endothelial cell damage. These findings provide new insights for understanding the underlying mechanism of estrogen replacement therapy. Further research is needed to further analyze the molecular mechanism of estrogen enhancing the autophagy program and to conduct related animal experiments.

Acknowledgements

We thank all the women who participated in this study and the Hunan Provincial People’s Hospital for their support.

Author contributions

TongYu: research design, data analysis, and manuscript writing. xiuqinhong: funding acquisition, conceptualization, and writing—review and editing. YiYang: data collection and research design. Dandan Zhang: data collection and comments. Yu Jiang, Jia Wang, Jing Li, Yannan Zhang, Rong Xie: data collection and investigation. All authors contributed to the article and approved the submitted version.

Funding

This work was sponsored by grants from the National Natural Science Foundation of China (No.81773530, 81202281) and Natural Science Foundation of Hunan Province (No.2020JJ4047).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Hunan Normal University (No. 034/2017). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. All methods are carried out in accordance with the relevant guidelines and regulations.

Consent for publication

Get permission from all authors.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Tousoulis, D; Papageorgiou, N; Androulakis, E; Paroutoglou, K; Stefanadis, C. Novel therapeutic strategies targeting vascular endothelium in essential hypertension. Expert Opin. Investig. Drugs; 2010; 19, pp. 1395-1412.1:CAS:528:DC%2BC3cXhtlWmurfE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20923260]

2. Sebastian, A; Cordain, L; Frassetto, L; Banerjee, T; Morris, RC. Postulating the major environmental condition resulting in the expression of essential hypertension and its associated cardiovascular diseases: dietary imprudence in daily selection of foods in respect of their potassium and sodium content resulting in oxidative stress-induced dysfunction of the vascular endothelium, vascular smooth muscle, and perivascular tissues. Med. Hypotheses; 2018; 119, pp. 110-119.1:CAS:528:DC%2BC1cXhsVyns7vF [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30122481]

3. Feng, Y et al. Value of plasma homocysteine to predict stroke, cardiovascular diseases, and new-onset hypertension: A retrospective cohort study. Med. (Baltim).; 2020; 99, e21541.

4. Sato, K; Nishii, T; Sato, A; Tatsunami, R. Autophagy activation is required for homocysteine-induced apoptosis in bovine aorta endothelial cells. Heliyon; 2020; 6, e03315.1:CAS:528:DC%2BB2MXmt1Klsro%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32021943][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994847]

5. Du, J; Zhang, C; Zhao, W. Autophagy and hypertension. Adv. Exp. Med. Biol.; 2020; 1207, pp. 213-216.1:CAS:528:DC%2BB3MXhtVKjs7rP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32671749]

6. Mizushima, N; Komatsu, M. Autophagy: renovation of cells and tissues. Cell; 2011; 147, pp. 728-741.1:CAS:528:DC%2BC3MXhsVKgsLnN [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22078875]

7. Chen, K; Sun, Z. Autophagy plays a critical role in Klotho gene deficiency-induced arterial stiffening and hypertension. J. Mol. Med. (Berl); 2019; 97, pp. 1615-1625.1:CAS:528:DC%2BC1MXhvF2gt73K [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31630227]

8. Zhang, Y et al. Oxymatrine inhibits Homocysteine-Mediated autophagy via mif/mtor signaling in human umbilical vein endothelial cells. Cell. Physiol. Biochem.; 2018; 45, pp. 1893-1903.2018pgrb.book...Z1:CAS:528:DC%2BC1cXms1aqu70%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29510402]

9. Tian, X et al. MicroRNA-199a-5p aggravates primary hypertension by damaging vascular endothelial cells through Inhibition of autophagy and promotion of apoptosis. Exp. Ther. Med.; 2018; 16, pp. 595-602. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30116316][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090226]

10. Blenck, CL; Harvey, PA; Reckelhoff, JF; Leinwand, LA. The importance of biological sex and Estrogen in rodent models of cardiovascular health and disease. Circ. Res.; 2016; 118, pp. 1294-1312.1:CAS:528:DC%2BC28XmtVaht7o%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27081111][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834858]

11. Tao, Z et al. Estradiol Signal. Mediates Gend. Difference Visc. Adiposity Via Autophagy; 2018; 9, 309.

12. Wang, F; Xiao, J; Shen, Y; Yao, F; Chen, Y. Estrogen protects cardiomyocytes against lipopolysaccharide by inhibiting autophagy. Mol. Med. Rep.; 2014; 10, pp. 1509-1512. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25017426]

13. Kawagoe, J et al. Mechanism of the divergent effects of Estrogen on the cell proliferation of human umbilical endothelial versus aortic smooth muscle cells. Endocrinology; 2007; 148, pp. 6092-6099.1:CAS:528:DC%2BD2sXhsVers7rO [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17872375]

14. Zhang, Y et al. 17β-estradiol attenuates homocysteine-induced oxidative stress and inflammatory response as well as MAPKs cascade via activating PI3-K/Akt signal transduction pathway in Raw 264.7 cells. Acta Biochim. Biophys. Sin (Shanghai); 2015; 47, pp. 65-72. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25605419]

15. Xiang X, Huang J, Song S, Wang Y, Zeng Y, Wu S, Ruan Y. 17β-estradiol inhibits H2O2-induced senescence in HUVEC cells through upregulating SIRT3 expression and promoting autophagy. Biogerontology. 2020 Oct;21(5):549-557. https://doi.org/10.1007/s10522-020-09868-w. Epub 2020 Mar 14. PMID: 32172411.

16. Perrotta, I. The use of electron microscopy for the detection of autophagy in human atherosclerosis. Micron; 2013; 50, pp. 7-13. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23623713]

17. Ou, H et al. Effect of nuclear actin on endothelial nitric oxide synthase expression. Arterioscler. Thromb. Vasc Biol.; 2005; 25, pp. 2509-2514.1:CAS:528:DC%2BD2MXht1Crt7zM [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16210567][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1382336]

18. Onuh, JO; Qiu, H. New progress on the study of aortic stiffness in age-related hypertension. J. Hypertens.; 2020; 38, pp. 1871-1877.1:CAS:528:DC%2BB3cXhslClt77P [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32890259][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647939]

19. Derzko, C. M., Röhrich, S. & Panay, N. Does age at the start of treatment for vaginal atrophy predict response to vaginal estrogen therapy? Post hoc analysis of data from a randomized clinical trial involving 205 women treated with 10 µg estradiol vaginal tablets, Menopause (2020).

20. Wang, F et al. The relationship between plasma homocysteine levels and MTHFR gene variation, age, and sex in Northeast China. Niger J. Clin. Pract.; 2019; 22, pp. 380-385.2019ccce.book...W1:STN:280:DC%2BB3cbgslOltA%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30837427]

21. Ahmad, H. et al. Red Cell Distribution Width Is Positively Correlated with Atherosclerotic Cardiovascular Disease 10-Year Risk Score, Age, and CRP in Spondyloarthritis with Axial or Peripheral Disease, (2018) 2476239. (2018).

22. Zhang D, Hong X, Wang J, Jiang Y, Zhang Y, Chen J, Niu X. Estradiol-17β inhibits homocysteine mediated damage by promoting H2 S production via upregulating CBS and CSE expression in human umbilical vein endothelial cells. J Cell Biochem. 2021 Sep;122(9):915-925. 10.1002/jcb.29527. Epub 2019 Nov 14. PMID: 31724756.

23. Madej, A; Dąbek, J; Majewski, M; Szuta, J. Effect of Perindopril and Bisoprolol on IL-2, INF-γ, hs-CRP and T-cell stimulation and correlations with blood pressure in mild and moderate hypertension. Int. J. Clin. Pharmacol. Ther.; 2018; 56, pp. 393-399.1:CAS:528:DC%2BC1MXjt1KqsLs%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29809132]

24. Hansson, GK; Libby, P. The immune response in atherosclerosis: a double-edged sword. Nat. Rev. Immunol.; 2006; 6, pp. 508-519.1:CAS:528:DC%2BD28XmtFegsLc%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16778830]

25. Libby, P; Hansson, GK. Inflammation and immunity in diseases of the arterial tree: players and layers. Circ. Res.; 2015; 116, pp. 307-311.1:CAS:528:DC%2BC2MXpslKhsw%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25593275][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299915]

26. Srivaratharajah, K; Abramson, BL. Hypertension in menopausal women: the effect and role of Estrogen. Menopause; 2019; 26, pp. 428-430. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30889094]

27. Shapiro, S; Pines, A. Menopausal hormone therapy and risk of hypertension. Climacteric; 2012; 15, 635. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23316488]

28. Mounier-Vehier, C; Angoulvant, T; Boivin, JM; Plu-Bureau, G. [Hypertension and menopausal hormone therapy]. Presse Med.; 2019; 48, pp. 1295-1300.1:STN:280:DC%2BB3MfgtVWgsw%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31735524]

29. Libby, P; Ridker, PM; Hansson, GK. Progress and challenges in translating the biology of atherosclerosis. Nature; 2011; 473, pp. 317-325.2011Natur.473.317L1:CAS:528:DC%2BC3MXmtlemu7w%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21593864]

30. Ren, H et al. Selenium inhibits Homocysteine-Induced endothelial dysfunction and apoptosis via activation of AKT. Cell. Physiol. Biochem.; 2016; 38, pp. 871-882.1:CAS:528:DC%2BC28XltVansrw%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26909517]

31. Gurda, D; Handschuh, L; Kotkowiak, W; Jakubowski, H. Homocysteine Thiolactone and N-homocysteinylated protein induce pro-atherogenic changes in gene expression in human vascular endothelial cells. Amino Acids; 2015; 47, pp. 1319-1339.1:CAS:528:DC%2BC2MXltVSnsrw%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25802182][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458266]

32. Almashhadany, A et al. Homocysteine exposure impairs myocardial resistance to ischaemia reperfusion and oxidative stress. Cell. Physiol. Biochem.; 2015; 37, pp. 2265-2274.1:CAS:528:DC%2BC28XksFWksg%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26624798]

33. Martinet, W; De Meyer, GR. Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ. Res.; 2009; 104, pp. 304-317.1:CAS:528:DC%2BD1MXhsV2jsLk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19213965]

34. Das G, Shravage BV, Baehrecke EH. Regulation and function of autophagy during cell survival and cell death. Cold Spring Harb Perspect Biol. 2012 Jun 1;4(6):a008813. https://doi.org/10.1101/cshperspect.a008813. PMID: 22661635; PMCID: PMC3367545.

35. Park, J; Shin, H; Song, H; Lim, HJ. Autophagic regulation in steroid hormone-responsive systems. Steroids; 2016; 115, pp. 177-181.1:CAS:528:DC%2BC28XhsFamtb3L [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27643453]

36. Schneider, JL; Cuervo, AM. Autophagy and human disease: emerging themes. Curr. Opin. Genet. Dev.; 2014; 26, pp. 16-23.1:CAS:528:DC%2BC2cXht12nu7rI [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24907664]

37. Chung MT, Lee YM, Shen HH, Cheng PY, Huang YC, Lin YJ, Huang YY, Lam KK. Activation of autophagy is involved in the protective effect of 17β-oestradiol on endotoxaemia-induced multiple organ dysfunction in ovariectomized rats. J Cell Mol Med. 2017 Dec;21(12):3705-3717. 10.1111/jcmm.13280. Epub 2017 Jul 17. PMID: 28714586; PMCID: PMC5706505.

38. Che, J et al. Kaempferol alleviates ox-LDL-induced apoptosis by up-regulation of autophagy via inhibiting PI3K/Akt/mTOR pathway in human endothelial cells. Cardiovasc. Pathol.; 2017; 31, pp. 57-62.1:CAS:528:DC%2BC2sXhs1SitLfP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28985493]

39. Knoka, E et al. Circulating plasma microRNA-126, microRNA-145, and microRNA-155 and their association with atherosclerotic plaque characteristics. J. Clin. Transl Res.; 2020; 5, pp. 60-67. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32377580][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197049]

40. Xiao-Hong, D; Chang-Qin, X; Jian-Hua, H; Wen-Jiang, Z; Bing, S. Icariin delays homocysteine-induced endothelial cellular senescence involving activation of the PI3K/AKT-eNOS signaling pathway. Pharm. Biol.; 2013; 51, pp. 433-440. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23336586]