Rutin Reverses Abnormal Autophagy in Hypoxic Pulmonary Arterial Hypertension via Regulation of Mitofusin 1


 Background:Recently, rutin, a citrus flavonoid occurring in many plants, has been found to be beneficial in preventing hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) from proliferating via scavenged reactive oxygen species (ROS). However, rutin’s underlying mechanism of action against PASMCs in the context of hypoxia is still unclear. Autophagy, the main intracellular degradation and recycling process, exerts a critical adaptive effect on the pathological angiogenesis associated with hypoxic pulmonary arterial hypertension (HPAH) by removing damaged mitochondria and regulating ROS production and cell proliferation. It would be useful to identify the role of rutin and its interaction with autophagy in exerting protective effects against HPAH.Methods:We chose 21 SD rats and randomly and equally divided them into three groups of normoxia, hypoxia, and hypoxia + rutin. At the end of the exposure period, we measured the right ventricular systolic pressure (RVSP), the weight of right ventricle (RV), and the ratio of RV weight to left ventricular (LV) weight plus septum (RV/LV+S) of each rat. PASMCs of the three groups of rats were isolated and cultured, and the effect of rutin on autophagy-related protein expression under hypoxia was analyzed using immunofluorescence analysis, transmission electron microscopy, western blot (WB) analysis, and siRNA design and transfection.Results:We found RVSP, RV/LV+S, and pulmonary artery wall thickness were reduced by rutin in the pulmonary arterial hypertension (PAH) animal model. WB results showed that rutin regulated expression of autophagy-related proteins. Moreover, rutin downregulated Mitofusin 1 (Mfn1) over-expression induced by hypoxia. But when Mfn1 was silenced, there was little difference in the expressions of beclin-1 (BECN-1), and other marker proteins with or without rutin.Conclusions:Rutin suppressed the abnormal autophagy of hypoxia-induced PASMCs via the regulation of the target, Mfn1. This revealed the protective effect of rutin on vascular remodeling caused by hypoxia and demonstrated how rutin could slow down the development of HPAH.


Introduction
Pulmonary arterial hypertension (PAH) is a rare and malignant cardiovascular disease (CVD) characterized by continuous increases in mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR) that lead to heart failure [1][2][3]. The average patient's survival time from diagnosis to death is only 2.8 years [3]. Moreover, pulmonary arterial disorders and myocardial lesions caused by PAH are impossible to cure by conventional therapeutics. Based on this, it is universally acknowledged that identifying an effective treatment for PAH is urgent. Traditional Chinese medicine (TCM) approaches the treatment of various diseases using "multi-target" techniques, which are effective and safe [4]. Hence, TCM has great advantages and developmental prospects [5]. In recent years, studies in TCM has provided many drug candidates that are expected to cure PAH [6].
Nonetheless, it remains unclear whether rutin has therapeutic effects on PAH, and what are the underlying mechanisms leading to rutin's mechanism of e cacy.
Mitochondria, as hallmarks of eukaryotic cells, are signi cant sites for aerobic respiration and the main sites for synthesizing and producing ROS [19]. Thus, mitochondria play a crucial role in the development of cell biology and multiple diseases. As for mammals, the morphological changes of mitochondria are achieved by fusion and division. The fusion/division of mitochondria depends on Mitofusin 1/2 (Mfn1/2), mitochondrial dynamin-like GTPase (OPA1), and dynamin-related protein (Drp1), which consists of a GTP functional domain and a crimp-crimp domain [20][21]. Under hypoxic conditions, mitochondrial imbalance leads to a large release of ROS that induce morphological changes in the mitochondria, which, in turn, trigger a series of cellular effects, such as the abnormal occurrence of autophagy, apoptosis, and proliferation [22].
In the absence of nutrients, autophagy can help cells survive, but abnormal autophagy may lead to the occurrence of a variety of human diseases, such as obstructive pulmonary disease and idiopathic pulmonary brosis [28][29][30]. There have been many reports that the abnormal occurrence of autophagy can lead to the pathological angiogenesis of PAH [24][25][26][27][28][29][30][31], and ROS can cause abnormal autophagy under pathological conditions [32]. In this paper, we explored the effects and the molecular mechanism of action of rutin on hypoxic pulmonary artery hypertension (HPAH). Additionally, we also screened for the key protein target that could contribute to rutin's regulation of HPAH.

Animal model
Five-to six-week-old adult male SD rats (250 to 300 g) were obtained from the Experimental Animal Center in our university. Twenty-one SD rats were randomly and equally divided into three groups of normoxia, hypoxia, and hypoxia + rutin. All rats were provided with su cient food and water. The 7 rats in the normoxic group were kept in a normoxic environment with a FiO 2 of 0.21 for 21 days. The other fourteen rats in the hypoxic (7 rats) and rutin (7 rats) groups were housed in a hypoxic environment with a FiO 2 of 0.12 for 21 days [33]. At the beginning of the exposure period, the rats in the rutin group were randomly induced with an intraperitoneal injection of rutin, which was dissolved in 5% cellulose sodium carboxymethyl (CMC) at a dose of 200 mgkg -1 ⋅d -1 for 21 days. The other rats in the normoxic and hypoxic groups were only induced by 5% CMC on the same days.

RVSP and ventricular weight measurements in rats
At the end of the exposure period, the RVSP of each rat was measured by right heart catheterization. Then, the animals were euthanized by decapitation. The rat hearts were quickly excised, the free wall of the RV was dissected, and each chamber was weighed. The (RV/LV+S) ratio was used as an index of RV hypertrophy. Finally, lung tissue was extracted and retained for subsequent experiments.

Morphological analysis of the pulmonary artery in rats
The rat lungs were washed with 1 × phosphate-buffered saline (PBS) and stored in the freezer at -80 °C. The remaining lung tissue was immersed in 4% paraformaldehyde, and then embedded in para n wax and cut into sections. The sections were stained with hematoxylin-eosin (H&E) staining (AR1180, Boster).
The lung tissue sections were examined by digital photomicrography and analyzed with Image-Pro Plus 6.0.

Cell isolation and culture
PASMCs were prepared from rat pulmonary arterial (PA) tissue. The PA was slit to remove the pulmonary artery endothelial cells (PAECs) and washed with 1 × HEPES buffer. The PASMCs were digested by using 1 mg/mL collagenase II at 37 °C for 30 min and were washed once with 1 × phosphate buffered saline (PBS) to remove the collagenase II. Then, PASMCs were cultured in Dulbecco's modi ed Eagle's medium (DMEM) (SH30022.01B, Hyclone) with 20% fetal bovine serum (FBS) (SV30087.03, Hyclone) and incubated at 37 °C. Finally, the cell culture medium was replaced, and the cells were ready to use in subsequent experiments. In vitro PASMCs were treated with 0.1 μmol/L rutin and exposed to hypoxia for 24 h by placing the cells in a hypoxia chamber (THERMO) with a water-saturated atmosphere comprising 3% O 2 , 5% CO 2 , and 91% N 2 .

Transmission electron microscopy
The PASMCs centrifuged at 3000 × g for 15 min and xed with 2% glutaraldehyde (electron microscopy grade), and the photographing of autophagic vacuoles was consigned to the Electron Microscopy Center of our university.

Statistics
The data are presented as the mean ± standard deviation (SD). The analysis was performed with Student's t-test or one-way analysis (ANOVA) by Dunnett's test, where appropriate. The statistical signi cance level was set at P < 0.05. Statistical analysis was performed with Statistical Product and Service Solutions version 13.0.

Rutin attenuated the development of HPAH in the rat model
To observe the effect of rutin on the development of PAH, the rats received intragastric administration of rutin during the preparation of HPAH rats. Then, we characterized the PH parameters in detail, mainly including the indirect index of cardiac function (RVSP and RV/LV + S ratio) and vascular remodeling. As shown in Fig. 1 (A, B), hypoxia signi cantly elevated the RVSP and RV/LV + S ratio, while this impact was reversed by rutin. In addition, H&E staining showed that the ratio of the intimal-to-medial area was signi cantly increased in HPAH models, and rutin reversed the wall thickening induced by hypoxia (Fig.   1C). Taken together, these results indicate that rutin prevented the pathological development of pulmonary arterial hypertension.

Rutin affected the expression of BECN-1 in lung tissue and PASMCs
According to reports, hypoxia-induced autophagy is a characteristic of HPAH [25]. Therefore, we investigated whether rutin protected against HPAH through inhibiting the autophagy of PASMCs in rats.
We observed the changes of the autophagy-related protein BECN-1 with cell immuno uorescence, WB, and immunohistochemical experiments. As shown in Fig. 2 (A), the immuno uorescence staining displayed that hypoxia led to up-expression of BECN-1, but rutin reversed over-expression of BECN-1. Furthermore, we found the autophagy occurred in the smooth muscle cell layer under hypoxic conditions, and rutin could reverse abnormal autophagy. WB results indicated the expression of BECN-1 in hypoxic lung tissue and PASMCs was signi cantly up-regulated, however, the expression of BECN-1 was downregulated when the rats and PASMCs under hypoxia were pre-treated with rutin, shown in Fig. 2 (B, C). At the same time, immunohistochemical analysis displayed the expression of BECN-1 in lung tissues, and we discovered that rutin could decrease the expression of BECN-1 under hypoxic conditions (Fig. 2D).
Thus, our results show that rutin could induce a decrease in BECN-1 expression under hypoxia.

Rutin reduced occurrence of autophagy-induced chronic hypoxia
To investigate molecular mechanism of rutin affecting autophagy of PASMCs, we used an electron microscope to analyze the autophagy morphology. There were immature autophagy vacuoles (AVi) and degradative autophagic vacuoles (AVd) in PASMCs under hypoxia, which are usually generated during the development of autophagy. But we observed few AVi and AVd in the PASMCs pretreated with rutin under hypoxia (Fig. 3A). Then, we performed the plasmid eGFP-mRFP-LC3 uorescence assay. Compared with the normoxic group, the autolysosomes (yellow dots) and autophagosomes (red dots) in the hypoxia group were signi cantly increased, however; rutin decreased the numbers of autolysosomes and autophagosomes (Fig. 3B). WB was used to detect the expression of autophagy-related proteins, such as sequestosome 1 (SQSTM1) in lung tissues and PASMCs. As shown in Fig. 3 (C, D), rutin signi cantly down-regulated the over-expression of ATG5 and ATG7, and up-regulated low-expression of SQSTM1 induced by hypoxia both in the rats and PASMCs. These ndings suggest that rutin could inactivate the autophagy process in PASMCs under hypoxic conditions.

Mfn1 played a key role in the process of rutin suppressing PASMCs abnormal autophagy
It has been reported that Mfn1 in uences the mitochondrial dynamics involved in hypoxia-induced proliferation in PASMCs [35]. Therefore, we explored the changes in Mfn1 expression in rat lung tissue and PASMCs induced by hypoxia. We found that Mfn1 expression was up-regulated under hypoxic conditions, while rutin suppressed Mfn1 over-expression induced by hypoxia (Fig. 4). Then, we used silencing Mfn1 with siRNA transfection to verify our ndings, and the transfection e ciency is shown in Fig. 5A. The WB experiment was used to observe the effects of rutin on the expression levels of the proteins ATG5, ATG7, BECN-1, and SQSTM1 after silencing Mfn1 with siRNA. The expression of ATG5, ATG7, and BECN-1 in the hypoxic group (silenced Mfn1 with siRNA) were down-regulated and SQSTM1 was up-regulated. A similar effect was obtained in the hypoxic group treated by rutin. More importantly, the hypoxic group with siMfn1 and rutin did not show further inhibition of hypoxia-induced abnormal expression of autophagy-related proteins (Fig. 5B,C). These results demonstrate that Mfn1 played a prominent role in rutin regulating autophagy in PASMCs under hypoxia.

Discussion
In this study, we provided the rst evidence of the protective molecular mechanisms of rutin in PAH and PASMCs under hypoxia in an animal model. The novel ndings in this paper consist of two points. Firstly, rutin alleviated hypoxia-induced pulmonary vascular remodeling and prevented the development of pulmonary hypertension. Secondly, rutin reduced the occurrence of hypoxia-induced autophagy in PASMCs. Mfn1 played a crucial role in this process, which indicates that rutin inhibited PASMCs autophagy by regulating the expression of Mfn1.
PAH is a malignant cardiovascular disease with poor prognosis and a high fatality rate [36][37]. Recently, PAH treatment has consisted of systemic vasodilatation drugs, which lack speci city to pulmonary circulation. Moreover, these drugs have strong side effects, undermining the treatment effect and causing great harm to the human body. Seeking safe and effective treatments, researchers have turned to TCM. Rutin has many pharmacological activities, such as strengthening and regulating the permeability of blood vessel walls [38]. Based on this, we hypothesized that rutin could have a certain inhibitory effect on the progress of HPAH. Through establishing an HPAH rat model, we observed the effects of rutin on right ventricular function in HPAH rats. We found that rutin reduced the increased RVSP and RV/LV + S induced by hypoxia, which proved that rutin improved the right ventricular function in the setting of HPAH. Moreover, H&E staining results indicated that rutin could also delay pulmonary vascular remodeling.
These results suggest that rutin had a preventive effect on HPAH. However, the speci c mechanism involved in the process of HPAH prevented by rutin remains unclear.
Autophagy is a vital process in maintaining cellular homeostasis and survival. Many reports have shown that autophagy could affect the progression of PAH [31]. Based on this, we explored the effects of rutin on autophagy in rats with HPAH and PASMCs. Initially, using immuno uorescence and immunohistochemistry experiments, we found the application of rutin could effectively reduce the production of autophagy, which occurs in the smooth muscle cell layer under hypoxic conditions. Next, we employed WB analysis to detect the change in the autophagy marker protein expressions, such as ATG5, ATG7, BECN-1, and SQSTM1 [24]. The results showed the over-expression of ATG5, ATG7, and BECN-1 as well as the down-expression of SQSTM1 (under hypoxic conditions) were effectively reversed by rutin. In addition, immuno uorescence, electron microscope, and eGFPmRFP-LC3 plasmid transfection experiments were administered to observe immature autophagosomes and degraded autophagosomes in PASMCs. We discovered autolysosomes and autophagosomes returned to their normal levels in PASMCs pre-treated with rutin under hypoxia. All these results suggest that rutin inhibited the abnormal autophagy of PASMCs induced by hypoxia, which further veri ed that rutin attenuated the remodeling of pulmonary arteries by suppressing the occurrence of autophagy in PASMCs.
However, the question remains as to what molecular mechanism was responsible for the regulation of autophagy by rutin in PASMCs. In our previous work, we discovered that rutin, as an antioxidant, exhibited signi cant ROS scavenging properties, with ROS mainly generated by mitochondria under hypoxic conditions [18]. Meanwhile, the balance of mitochondrial fusion and ssion normalizes mitochondrial networks when the loss of homeostasis would lead to mitochondrial dysfunction. This has long been linked to the pathogenesis of neurodegenerative diseases, diabetes, myopathies, and many other human diseases. Recently, mitochondrial fusion and ssion have been linked to both mitophagy (mitochondrial autophagy) and global autophagy in mouse embryonic broblasts, human broblasts, and cardiac cells. One group reviewed fusion was brief and triggers ssion. Following a ssion event, the daughter mitochondrion may either maintain intact membrane potential, or depolarize. In the case mitochondrial depolarization is transient and Δψm resumed, fusion capacity was restored. However, if Δψm depolarization is sustained, elimination by autophagy occurs [10]. And other observations showed Mfn1 may affect occurrence of autophagy both mitophagy (mitochondrial autophagy) and global autophagy, that is to sequester portions of cytosol or damaged and surplus organelles into autophagosomes and degrade them in autolysosomes, some signaling pathways, such as NFKB and TP53 involved in these process [11][12][13][14]. However, it is still unclear whether the balance of mitochondrial fusion and ssion induces autophagy, and what their combined effects are on apoptosis or proliferation. Our previous work showed rutin reduced the production of ROS to reverse the abnormal proliferation induced by hypoxia [15], so it will be of interest to explore whether Mfn1 is a key target protein in the process of rutin regulation of abnormal autophagy. Based on this, in our current study, we used WB to observe that upexpression of the altered autophagy markers under hypoxia were inhibited by rutin, which could be regulated by siMfn1 to further to verify our hypothesis. Thus, these ndings suggest that rutin could decrease the anomalous expression of Mfn1 induced by hypoxia, which alleviated the abnormal autophagy in PASMCs under hypoxia.
In the present study, we focused on exploring rutin's bioactivity, and its mechanism of action in the setting of HPAH. Because of its chemical structure, rutin solubility in water is poor leading to low bioavailability in vivo. Today, we have little knowledge about the potential use of this compound in patients with HPAH. We face the problem of how to improve rutin solubility in water, decrease its dose administration to humans and increase its possible applications in clinical treatment. Thus, our next work will explore rutin's pharmacodynamics (how rutin is metabolized and how to increase its bioavailability in vivo) so that rutin can be used as a treatment in a clinical setting.

Conclusions
In conclusion, our study elucidates the protective role of rutin and how it slowed progression of HPAH in rats. The underlying mechanism contributing to this nding was that rutin attenuated the abnormal autophagy of PASMCs under hypoxia and HPAH in rats. We discovered the mitochondria-Mfn1-ROS pathway involved in this process, especially Mfn1 regulated by rutin as a target protein, played a key role in rutin's suppression of the remodeling of pulmonary arteries that ultimately slowed the development of HPAH in rats. Our study will help to deepen our understanding of HPAH treatment and guide us to

Declarations
Ethics approval and consent to participate All experimental procedures in animals which were approved by the Institutional Animal Care and Use Committee were carried out in accordance with guidelines for the Care and Use of Laboratory Animals, and it was conducted in compliance with the NIH guide. The study protocol in the Ethics of Animals was approved by the Committee Experiments of the Harbin Medical University (Permit Number: 2010-0006). All surgeries were carried out under sodium pentobarbital anesthesia, and all efforts were made to minimize pain.

Consent for publication
Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article.