Zhengyuan prescription inhibits X-ray-induced injury of human umbilical vein endothelial cells by activating the Nrf2 signaling pathway

Background: Zhengyuan prescription (ZYP) is a Chinese herbal medicine used in clinical practice to protect against radiotherapy-induced injuries. In this study, we investigate the protective effect of ZYP against X-ray-induced injury of human umbilical vein endothelial cells (HUVECs), and we explore the mechanisms underlying this effect. Methods: After 3 h of ZYP intervention, the cells in the ZYP group were irradiated with 6 Gy X-rays and cultured for 48 h. Subsequently, the cell viability, cell morphology, mitochondrial membrane potential, and apoptosis and oxidative stress markers were observed, as well as the expressions of apoptotic and oxidative stress proteins. Results: The obtained results demonstrate that exposure to X-rays promotes cell death, reduces mitochondrial membrane potential, and induces the pirroduction of intracellular reactive oxygen species (ROS). Pretreatment with ZYP reverses these effects to a great extent. Moreover, it up-regulates the expression of the B-cell lymphoma 2 (Bcl-2) apoptosis inhibitor protein while down-regulating the expressions of Bcl-2-associated X protein (Bax), caspase-3, and caspase-9. Interestingly, ZYP can also inhibit oxidative stress injury by activating the expression of Nrf2 (Nuclear Factor E2 related factor) Conclusions: This study is the rst to demonstrate that ZYP suppresses X-ray-induced injury of HUVECs by activating the Nrf2 signaling


Background
Radiotherapy is an e cient tumor treatment that can effectively prolong the lives of patients and enhance their quality of life [1,2]. In some cases, radiotherapy has resulted in therapeutic outcomes better than those achieved using other available treatments [3]. However, the application of radiotherapy treatment is limited by its side effects, including high toxicity and normal tissue damage [4,5]. The mechanisms underlying the development of these side effects implicate oxidative stress [6]. To minimize the oxidative imbalance effect associated with ionizing radiation and to improve the targeting effect of future radiotherapy treatments, the sensitivity of tumor cells towards ionizing radiation (IR) must be increased, and the effect of this radiation on normal tissues must be reduced [7].
Traditional Chinese medicine (TCM), especially Chinese herbal medicine, has evolved over thousands of years in China, Japan, and other Asian countries. TCM has been used in cancer prevention and treatment therapies, and it has shown good e ciency [8][9][10]. Moreover, preclinical and clinical studies have shown that the combination of TCM and conventional western medicine (chemotherapy and radiotherapy) can provide effective supportive care for cancer patients [8,11]. According to TCM theory, the acute injury caused by IR is related to the heat toxicity of the radiation, which consumes qi and yin upon entering the human body [12]. ZYP is a Chinese herbal prescription that is composed of Radix Angelicae Sinensis, Radix Panacis Quinquefolii, Herba Agrimoniae, Paris polyphylla and Adenophora tetraphylla ( Table 1) that may be used to alleviate the symptoms of radiotherapy-induced syndrome by replenishing qi, replenishing blood, clearing heat, and detoxicating [10,13]. In previous studies, we had shown that ZYP has signi cant protective effects against 60Co γ-ray induced injury in mice and against cyclophosphamide-induced myelosuppression in guinea pigs [14]. However, the effects and mechanisms of ZYP on X-ray-induced injury remain unclear.
Vascular endothelial cells in blood vessels are highly sensitive to radiation [15]. Normally, these cells regulate the growth of blood vessels, the adhesion and non-adhesion of blood cells, the relaxation and contraction of the vessels, and the balance of anticoagulants and procoagulants [16]. Therefore, they play an important role in maintaining the integrity of the vascular structure, regulating the ow of blood, mediating the in ammatory response, and responding to immunity [17][18][19]. According to the literature, IR exposure at a certain dose induces morphological changes in vascular endothelial cells, including nuclear enlargement, karyopycnosis, polykaryocyte formation, and decreased cell homogeneity [18,20]. Moreover, exposure to IR may lead to the activation of human umbilical vein endothelial cells (HUVECs), even in the absence of pathogens (i.e. aseptic in ammation). The exact stimulant causing HUVEC activation probably causes oxidative stress as well [15,19]. In addition to the aforementioned effects, IR exposure inhibits the antioxidant mechanisms of endothelial cells by promoting ROS production and by threatening the integrity and survival of normal surrounding cells [6]. In this study, we investigate the antioxidant effect of ZYP on X-ray-induced injury of HUVECs, and we analyze the underlying mechanisms.

HPLC analysis
The composition of the ve-herb ZYP prescription was veri ed by HPLC analysis. First, the prescription was boiled for 2 h then ltered. The extraction process was repeated twice, then the extract was concentrated and spray dried. The obtained powder was dissolved in 60% methanol, and the solution was subsequently analyzed by high performance liquid chromatography (HPLC). HPLC-diode array detector (HPLC-DAD) analysis was performed on an Agilent-1260 system coupled with a DAD detector. An Inert Sustain C18 column (150 mm×4.6 mm, 5 μm) was maintained at a column temperature of 30°C. The mobile phase, consisting of (A) acetonitrile and (B) 0.1% phosphoric acid, was applied in the following linear elution gradient: 0-20 min, 18 Haemek, Israe). The cells were cultured in an incubator at 37°C, under 5% CO 2 atmosphere, until 80-90% con uency was reached. To evaluate the effects of ZYP on X-ray induced damage, the cells were randomly divided into six groups: A, negative control group; B, X-ray model group; C, X-ray+200 µg/mL vitamin D 2 group; D, X-ray+20 µg/mL ZYP group; E, X-ray+50 µg/mL ZYP group; and F, X-ray+100 µg/mL ZYP group.

Ionizing radiation
The cultured cells were exposed to X-rays issued from an X-ray irradiation system (Elekta, Sweden) equipped with a medical linear accelerator. The samples were divided into six groups irradiated with 0, 4, 6, 8, 10, and 14 Gy ionizing radiation doses at the rate of 300 μ/min. Annexin V-FITC staining FITC-labeled phalloidin and DAPI staining were used to detect the cytoskeletal distribution of HUVECs and evaluate the speci c morphological changes in the nucleus that induce apoptosis. First, HUVECs were seeded in 6-well plates at the density of 10×10 4 cells/well, after 3 h of ZYP intervention, the cells in the ZYP group were irradiated with 6 Gy X-rays and cultured for 48 h. Afterwards, the cells were xed with 4% paraformaldehyde for 15 min, followed by treatment with 0.5% Triton X-100 for 5 min. Prior to the addition of the V-FITC working solution, the cells were washed twice with phosphate-buffered saline (PBS), then they were incubated with V-FITC in the dark at 37°C. Thirty minutes later, the cells were again washed twice with PBS, stained with 5 µg/mL DAPI in the dark at 37°C for 15 min, then analyzed using an inverted uorescence microscope. Representative digital images were acquired for analysis.
Calcein/PI cell assay FITC-labeled phalloidin and DAPI staining were used to detect the cytoskeletal distribution of HUVECs and evaluate the speci c morphological changes in the nucleus that induce apoptosis. First, HUVECs were seeded in 6-well plates at the density of 10×10 4 cells/well, after 3 h of ZYP intervention, the cells in the ZYP group were irradiated with 6 Gy X-rays and cultured for 48 h. Afterwards, the cells were xed with 4% paraformaldehyde for 15 min, followed by treatment with 0.5% Triton X-100 for 5 min. Prior to the addition of the V-FITC working solution, the cells were washed twice with phosphate-buffered saline (PBS), then they were incubated with V-FITC in the dark at 37°C. Thirty minutes later, the cells were again washed twice with PBS, stained with 5 µg/mL DAPI in the dark at 37°C for 15 min, then analyzed using an inverted uorescence microscope. Representative digital images were acquired for analysis.

Cell proliferation detection via EDU labeling
HUVECs were seeded in 6-well plates at the density of 10×10 4 cells/well and treated with positive sources at different concentrations for EDU labeling. Thirty-six hours after X-ray irradiation, the cells were xed and in ltrated, then they were incubated with the Click-iT reaction mixture for 30 min in the dark. Subsequently, the cells were washed twice with PBS and stained with 5 µg/mL Hoechset for 10 min in the dark. After washing again with PBS (twice), the cells were nally analyzed by inverted uorescence microscopy. Representative digital images were acquired for analysis.

Determination of mitochondrial membrane potential
HUVECs were cultured, induced X-ray damage and treated with ZYP for 48 h. The cells were incubated with 2 µM rhodamine 123 in the dark at 37°C for 30 min. Next, the cells were washed twice with serumfree medium then incubated in this medium at 37°C, in the dark. Sixty minutes later, the uorescence of cells was measured using an inverted uorescence microscope and the cell count and uorescence intensity were determined using the ImageJ software.

Flow cytometry
To assess the mitochondrial membrane potential, the cells were stained with rhodamine 123, then they were washed with PBS and analyzed by a ow cytometer using BD FACSuite v1.0.6.
Apoptosis was detected using the Annexin-V/PI assay kit (Absin Bioscience Inc). For this purpose, the treated cells were washed with pre-cooled PBS then resuspended in 300 µL 1×Binding Buffer.
Subsequently, the cells were stained with 5 µL Annexin V-FITC and 5 µL PI in the dark for 15 min. The apoptotic cells were then detected using ow cytometry, and the proportion (%) of each sub-population of cells was determined using the FlowJo 7.6 software.

Evaluation of LDH, MDA, and SOD levels
The MDA and SOD levels in seeded HUVECs (10×10 4 cells/well in a 6-well plate) were determined according to the thiobarbituric acid and xanthine oxidation methods, respectively. To determine the LDH level, the supernatant of the seeded cells was collected. A microplate reader was used to measure MDA, SOD, and LDH levels based on absorbance values.

Western blotting
Western blot analysis was used to assess changes in the protein expressions of HUVECs after ZYP treatment and X-ray irradiation. After treatment with ZYP, the cells were collected and lysed in RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.), and the protein concentration was quanti ed using the bicinchoninic acid (BCA) assay. Subsequently, the protein samples were loaded separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes. Incubate the membrane with blocking solution (5% skimmed milk) at room temperature for 1 h. After washing, incubate with the corresponding primary antibody (Table 2) overnight at 4°C. Incubated with a secondary antibody (Cell Signaling Technology) coupled with horseradish peroxidase. Positive antibody binding was then visualized by ECL detection and analyzed by the Image J software (BioRad, Hercules, CA, USA).

Statistical analysis
All of the statistical analyses were carried out using a GraphPad Prism V6 (GraphPad Software Inc., San Diego, CA). The data are expressed as the mean ± S.D. Statistically signi cant differences among groups were analyzed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison tests. P < 0:05 was considered statistically signi cant.

HPLC pro le of ZYP
The expression and distribution of F-actin in HUVECs were observed using a uorescence microscope.
The green and blue colors in Figure 3 correspond to F-actin and the nuclei, respectively. Based on the recorded images, the HUVECs in the X-ray group exhibit deformed F-actin, short pseudopodoid contraction, disordered micro lament arrangement, loose skeleton arrangement, poor directivity, coarse stress ber formation in the cytoplasm with nucleus shrinkage, and rupture, unlike the cells in the control group. The observed features are characteristic of apoptotic cells. Pretreatment with ZYP (20-100 μg/mL) moderates the morphological changes listed above ( Figure 3A). Compared to X-ray group cells, the cells in the ZYP group have fewer stress bers, and more F-actin is distributed on the cell membranes.
At higher concentrations of ZYP, the uorescence intensity increases (Figure 3B), and the cell edges revert to smooth with irregular polygons, similar to the cells in the control group ( Figure 3C).

Effect of ZYP on X-ray-induced changes in HUVEC membrane potential
Modi ed mitochondrial membrane potential is an early determinant of the mitochondrial apoptotic pathway. To determine the effect of ZYP on X-ray-induced HUVEC membrane potential, the uorescence of rhodamine-labelled cells was analyzed. The images presented in Figures 4A and 4C demonstrate that compared to the control group, the cells in the X-ray treatment group exhibit signi cantly weakened intensity of green uorescence (P < 0.001), which indicates that the mitochondrial membrane potential of these cells is reduced. ZYP treatment (20-100 µg/mL) partially alleviates the effect of X-ray exposure, as evidenced by the increased uorescence intensity of ZYP-treated cells compared to X-ray group cells (P < 0.001). As shown in Figures 4B and 4D, changes in uorescence intensity re ect changes in cell viability.
Overall, these ndings indicate that ZYP prevents cell damage by affecting the mitochondrial membrane potential.

Effect of ZYP on X-ray-induced HUVEC apoptosis
The effect of ZYP on X-ray-induced HUVEC apoptosis was assessed by staining the cells in different groups with the Calcein-AM/PI double staining reagent then observing them using a uorescence microscope. Upon entering the cytoplasm, Calcein-AM is hydrolyzed by esterase into green uorescing Calcein. Meanwhile, the nuclear staining dye PI does not penetrate the cell membrane of living cells. Instead, it passes through the disordered regions of dead cell membranes, and upon reaching the nucleus, and embeds into the DNA double helix of the dead cells, thereby producing red uorescence. The number of live and dead cells in different groups may thus be estimated based on the microscopic observation of green and red uorescence signals, respectively. The images shown in Figure 5A demonstrate that compared to the control group, green uorescence in the X-ray group is signi cantly reduced, whereas red uorescence is enhanced. However, the green uorescence intensity of cells in the ZYP group is higher than that detected for the X-ray group, and the red uorescence intensity is lower.
To con rm that ZYP has a protective effect against X-ray-induced HUVEC apoptosis, Annexin V/PI ow cytometry experiments were conducted. As shown in Figure 5B, the percentage of apoptotic cells in the Xray group (6 Gy) is 23.79%, which is much greater than that detected in the control group (2.34%). Pretreatment with 20 and 50 µg/mL ZYP effectively reduces the apoptosis rates to 14.92% and 9.32% (P < 0.001), respectively. The estimated total numbers of apoptotic cells in the early and late stages con rm that ZYP can protect HUVECs against X-Ray-induced apoptosis.

Effect of ZYP on ROS, malondialdehyde (MDA), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) formation in HUVECs
The exposure of endothelial cells to X-ray radiation results in aseptic in ammation, which is normally caused by oxidative stress. Knowing that oxidative stress and the pro-in ammatory process are triggered by excessive ROS (metabolites of intracellular oxidation-reduction reactions) formation, the effect of ZYP on the number of reactive ROS in X-ray-induced HUVECs was evaluated, based on assessments of the MDA, SOD, and LDH levels. As shown in Figure 6A, the SOD level detected in the X-ray group is signi cantly less than that determined for the control group (P < 0.001). Contrarily, the LDH and MDA levels in cells exposed to X-ray radiation are signi cantly higher than those corresponding to control group cells (P < 0.01), as is the uorescence intensity of ROS (P < 0.001) (Figures 6A, 6d, and 6E). Pretreatment with ZYP (20-100 µg/mL) reduces the uorescence intensity of ROS (P < 0.001), the LDH level, and the MDA level ( Figures 6B, 6D, and 6E), while increasing the content of SOD compared to the Xray group. This effect is concentration-dependent ( Figure 6C), and it indicates that ZYP has protective activity against the development of oxidative stress.

Effect of ZYP on the expression of apoptosis-related proteins in X-ray-induced HUVECs
The effect of ZYP pretreatment on the expression levels of the Bcl-2, Bax, caspase-9, and caspase-3 apoptosis-related proteins in X-ray-induced HUVECs was assessed. The obtained results indicate that Xray exposure signi cantly increases the expression of Bax, caspase-9, and caspase-3 proteins compared to the control group ( Figures 7C, 7d, and 7e), while decreasing the level of Bcl-2 ( Figure 7B). However, pretreatment with ZYP reverses these effects by inhibiting the X-ray-induced expression of Bax, caspase-3, and caspase-9, and by increasing the level of Bcl2 (1.25 and 1.43-fold at 50 and 100 µg/mL ZYP, respectively). These results con rm that ZYP's effect on X-Ray-induced HUVEC apoptosis is related to the mitochondrial pathway.
Effect of ZYP on Nrf2, HO-1, and NQO1 levels To further clarify the mechanism by which ZYP protects HUVECs against X-ray-induced injury, the expression levels of Nrf2, HO-1, and NQO1 in different groups were detected using western blot analyses.
As shown in Figure 8, these levels are lower in the X-ray group than in the control group, but they signi cantly increase upon pretreatment with ZYP (P < 0.05). This indicates that ZYP can protect HUVECs against X-ray-induced oxidative injury through Nrf2/HO-1/NQO1 signaling.

Discussion
Cancer is one of the leading causes of morbidity and mortality worldwide, and has become a major global health concern. The World Health Organization (WHO) predicts that by 2030 an estimated 21.4 million new cases of cancer and 13.2 million cancer deaths will occur annually around the world [12]. Surgery, chemotherapy, and radiotherapy are the major conventional cancer treatments currently available [8]. Despite the effectiveness of chemoradiotherapy in some cases, its application is limited by high toxicity, normal tissue damage, and other side effects [4]. According to previous studies, therapies based on traditional Chinese medicine (TCM) improve the body's immune system function and are characterized by reduced side effects and increased sensitivity compared to chemoradiotherapy [8].
ZYP is an herbal mixture that is formulated based on the principles of Shenmai Powder and Danggui Buxue Decoction for the treatment of qi and blood de ciency [10,13]. This TCM has been used clinically for more than 20 years, and it can effectively treat the qi and blood de ciency syndrome caused by radiotherapy and chemotherapy of malignant tumors [8]. Speci cally, ZYP strengthens the body's ability to resist damage, promotes blood generation, and inhibits bleeding. In this study, the ngerprint of the ZYP extract was established using high performance liquid chromatography (HPLC). The results show that the extract consists of ve major components (ferulic acid, ginsenoside Rb1, ginsenoside Rg1, polyglucoside VII, and ginkgolide) whose anti-radiation effects have been previously reported. Radix Panacis Quinquefolii improves lung-yin, clears away de ciency re, promotes uid production, and quenches thirst [21]. Moreover, it restores the antioxidant enzymatic activity, cytokine level, and hormone level affected by IR in mice [22]. Meanwhile, Radix Angelicae Sinensis promotes blood circulation, relieves pain, replenishes the blood, and regulates menstruation [21]. It also enhances caspase-dependent apoptosis by down-regulating survivin, and increases the radiosensitivity of H22 cells to 12 C 6+ heavy IR [23]. Ferulic acid, the main active ingredient in ZYP, has anti-tumor and anti-in ammatory effects, and it protects against IR-induced apoptosis and oxidation via the ERK pathway [24]. As for Agrimony, it exhibits hemostasis, blood generation, malaria interception, detoxi cation, anti-oxidation, and anti-tumor activities [21]. In addition, agrimony lactone, the active ingredient in Agrimony, inhibits the proliferation of the human gastric adenocarcinoma AGS cell line [25]. Paris Polyphylla Smith presents detoxi cation, pain relief, and immunity regulation effects [21], whereas Polyphyllin I suppresses human osteosarcoma growth by inactivating the Wnt/β-actenin pathway in vitro and in vivo [26]. Radix Adenophorae nourishes the yin, moistens dryness, clears lung heat, cools the liver, and nurtures the bloo [21]. Based on the theory of TCM and existing research studies, ZYF can protect against radiation-induced injury of HUVECs.
Exposure to IR may lead to excessive production of reactive oxygen species, such as O 2 -(superoxide radical), OH (hydroxyl radical), and H 2 O 2 (hydrogen peroxide), which in turn leads to increased intracellular oxidative stress [8]. The destruction of macromolecules, such as nucleic acids, proteins, and enzymes, under the effect of oxidative stress results in DNA chain rupture and reduced content and activity of anti-injury repair enzymes, as well as in changes in tissue morphology, metabolic function, and microcirculation [24,27]. In this study, we show that the X-ray exposure promotes the excessive generation of ROS in HUVECs, which impairs the antioxidant mechanisms in these cells. To alleviate the effect of X-ray radiation, excess ROS must be eliminated [8]. In the cellular environment, free radicals are mainly removed by antioxidant enzymes such as SOD. The SOD metalloenzyme is prevalent in many organisms, and it is characterized by oxygen free radical scavenging activity. MDA is a degradation product of lipid peroxidation, and its content is directly related to the degree of lipid peroxidation in cells, which indirectly re ects the extent of cell damage [28,29]. In the cellular environment, free radicals are mainly removed by antioxidant enzymes such as SOD. The SOD metalloenzyme is prevalent in many organisms, and it is characterized by oxygen free radical scavenging activity. MDA is a degradation product of lipid peroxidation, and its content is directly related to the degree of lipid peroxidation in cells, which indirectly re ects the extent of cell damage The mitochondrion is an essential organelle that maintains the normal functioning of cells and that is in turn maintained by △Ψm [30,31]. Oxidative stress induces mitochondrial dysfunction, resulting in the transport of cytochrome C from the mitochondrial membrane to the cytoplasm. This triggers the activation of caspase-3 and caspase-9 enzymes in HUVECs, both of which are key cell death substrates that promote apoptosis [32]. The results obtained herein demonstrate that ZYP can partially protect against X-ray-induced (6 Gy) mitochondrial damage by inhibiting the expression of intracellular caspase-3 and caspase-9 proteins. This effect is dependent on the concentration of ZYP. Knowing that the contents of Bcl-2 and Bax (pro-apoptotic protein) in the outer membrane of the mitochondrion affect mitochondrial permeability, and that increased permeability promotes the transfer of apoptotic signals, the levels of Bcl-2 and Bax proteins in HUVECs were also assessed [24]. The results indicate that ZYP pretreatment alleviates the damaging effect of X-ray exposure on the mitochondrial membrane by regulating the expressions of Bax and Bcl-2, in addition to caspase-3 and caspase-9.
The key mechanism implicated in cellular antioxidant activity is the Nrf2/HO-1/NQO1 signaling pathway [33]. Nrf2 is a redox-sensitive transcription factor that is directly activated by ROS [19,34]. Under normal circumstances, Nrf2 binds to Keap1(Kelch-like ECH-associated protein 1) and undergoes ubiquitinationdependent proteasome degradation. However, excessive ROS production (oxidative stress) induces a conformational change in Keap1 (modi cation of some sensor cysteines), which interferes with its binding to Nrf2.

Conclusion
This study establishes that ZYP can protect against X-ray-induced injury of HUVECs by mediating ROS scavenging, activating the Nrf2/HO-1/NQO1 signaling pathway, and stabilizing the mitochondrial membrane. This suggests that the X-ray-protection activity of ZYP is attributed to its antioxidative ability.
To con rm the protective effect of ZYP on bodies exposed to X-ray radiation, further studies must be conducted, particularly in vivo studies.  Table 2 Antibodies used for western blotting.