4,7-Didehydro-neophysalin B Protects Rat Lung Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Damage through Nrf2-Mediated Signaling Pathway

The administration of 4,7-didehydro-neophysalin B is expected to be a promising strategy for mitigating oxidative stress in respiratory diseases. This study was aimed at investigating the efficacy of 4,7-didehydro-neophysalin B for apoptosis resistance of rat lung epithelial cells (RLE-6TN) to oxidative stress and evaluating its underlying mechanism of action. The RLE-6TN cells treated with hydrogen peroxide (H2O2) were divided into five groups, and 4,7-didehydro-neophysalin B was administered into it. To evaluate its mechanism of action, the expression of oxidative stress and apoptotic proteins was investigated. 4,7-Didehydro-neophysalin B significantly inhibited H2O2-induced RLE-6TN cell damage. It also activated the Nrf2 signaling pathway which was evident from the increased transcription of antioxidant responsive of KLF9, NQO1, Keap-1, and HO-1. Nrf2 was found to be a potential target of 4,7-didehydro-neophysalin B. The protein levels of Bcl-2 and Bcl-xL were increased while Bax and p53 were decreased significantly. Flow cytometry showed that 4,7-didehydro-neophysalin B protected RLE-6TN cells from apoptosis and has improved the oxidative damage. This study provided a promising evidence that 4,7-didehydro-neophysalin B can be a therapeutic option for oxidative stress in respiratory diseases.


Introduction
The respiratory epithelial cell damage is a key indicator of respiratory diseases. Investigation of respiratory epithelial cell injury and its underlying mechanism of action and development of new drugs against it are crucial steps to prevent and treat respiratory diseases. Hydrogen peroxide (H 2 O 2) is the main reactive oxygen species (ROS) involved in the regulation of redox reactions in biological activities through specific protein targets [1]. Recently, oxidative stress is considered as a key risk factor for respiratory diseases [2]; evidences showed that the causative factors of diseases including mycoplasma pneumonia are also linked with the oxidative stress [3][4][5][6]. Therefore, the active compounds with antioxidative properties tend to be the potential agents for the prevention of respiratory diseases.
In recent years, the use of active natural compounds against pathological conditions has gained considerable recognition. 4,7-Didehydro-neophysalin B and Physalin B are biologically active substances extracted from Physalis alkekengi L. var. franchetii. It has been confirmed that Physalin B has various pharmacological effects such as antiinflammatory [7], antitumor [8], antibacterial [9], and immune regulation [10] verified by various bioassay both in vitro and in vivo. 4,7-Didehydro-neophysalin B is a new kind of Physalin B lacking two hydrogen atoms. There is evidence that Physalin B has efficient antioxidant activity [8]. However, the protective effect and mechanism of 4,7-didehydro-neophysalin B against H 2 O 2 -induced lung injury remained elusive.
Nuclear factor-erythroid 2-related factor-2 (Nrf2) has been identified as a key regulator of antioxidative, antiinflammatory, and conjugation/detoxification proteins including NAD(P)H, quinine oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1) [11]. After oxidative stress caused by H 2 O 2 or other factors, the Nrf2 pathway is rapidly activated to eliminate intracellular ROS generation, thereby attenuating DNA damage induced by H 2 O 2 to reduce risk of subsequent lung apoptosis [12].
Activation of antioxidative defense such as phase II enzyme expression is an effective way to protect cells against oxidative damage [13]. Nrf2 combined with antioxidant response element (ARE) system is one of the most important defensive signaling pathways to regulate the transcription activity of antioxidases [14]. The RLE-6TN cell line has been widely used to evaluate lung function due to their hypersensitivity to H 2 O 2 [15,16]. Although many bioactive compounds have been reported against oxidative stress, still there is no relevant study on the cytoprotective effect of 4,7-didehydro-neophysalin B on RLE-6TN cells.
In this study, H 2 O 2 was used to establish the basic oxidative damage model so that we can investigate the effect of 4,7-didehydro-neophysalin B on the oxidative stress, cell apoptosis, and the role of Nrf2 signaling pathway. This study will promote the development of nutraceutical and functional food from Physalis alkekengi L. var. franchetii or its extracts for reducing the risk of oxidative stress-induced lung injury.

Materials and Methods
2.1. Preparation of 4,7-Didehydro-neophysalin B. Physalin was isolated from the stems of Physalis alkekengi L. var. franchetii. The plant was identified by Dr. Qiongming Xu from the College of Pharmaceutical Science, Soochow University. Physalin was purified, and the purity of Physalin is 72.39% by chromatography [17]. Physalin from the above reaction was pulverized, extracted with ethanol, loaded on a silica gel column, eluted, and purified with ethyl acetate. The obtained contents of 4,7-didehydro-neophysalin B were detected by chromatography.    3 Oxidative Medicine and Cellular Longevity control to determine whether Nrf2's siRNA was successfully transfected.

Detection of Gene Expression in Tissues by RT-PCR.
The total RNA was extracted from cells using TRIzol reagent, and the absorbance was measured at 260 and 280 nm by an ultraviolet spectrophotometer. The RNA content was calculated from the absorbance (A) value at a wavelength of 260 nm while the RNA purity was identified by the ratio of A 260 /A 280 . Reverse transcription of total RNA was performed with reverse transcriptase. The PCR primers (shown in Table 2) were designed and synthesized by KeyGEN Bio-TECH with GAPDH as endogenous control, and the PCR reactions were carried out according to the recommended conditions.
2.6. Flow Cytometry. After treating the cells with/without 100 μmol of H 2 O 2 for 12 h, different concentrations of 4,7didehydro-neophysalin B (0, 2.5, 5, and 10 μg/mL) were added to the rat lung epithelial cells RLE-6TN for 24 h. The cells were collected, washed twice with cold PBS, and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were made into a single cell suspension. 5 μL of Annexin V•FITC and 5 μL PI were added, mixed, and incubated for 15 min in the dark. Finally, 400 μL of binding buffer was added and cell apoptosis was detected using flow cytometry.
To further explain the molecular mechanism by which 4,7-didehydro-neophysalin B treated H 2 O 2 -induced oxidative damage in RLE-6TN cells, western blot was performed. Nrf2's siRNA was generated to inhibit Nrf2 expression, and the inhibitory efficiency was verified using western blot. Nrf2's siRNA was transfected into RLE-6TN cells with or without H 2 O 2 treatment. Then, 10 μg/mL 4,7-didehydroneophysalin B was administrated for treatment and the expression of Nrf2 was explored using western blotting.
2.8. Statistical Analysis. Statistical analysis was performed by one-way analysis of variance with Tukey's test post hoc comparisons and Student's t-test when comparing between 2 groups using SPSS 19.0 software (USA). The data were presented as the mean ± SEM. Values with p < 0:05 was considered statistically significant.   Figure 3(b). Therefore, 12 h (68:88 ± 0:95%, p = 0:0002 < 0:001) was selected as a working time.

Knockdown of Nrf2
Declines the Treatment Effect of Physalin B. The Nrf2's siRNA treatment significantly reduced the expression of Nrf2 as shown in Figure 7. Compared with the model group, the treatment effect of 4,7-didehydro-neophysalin B in the Nrf2's siRNA treatment group was significantly reduced.

Discussion
A variety of chemicals and environmental factors causes oxidative stress in the human body which damages and induces a variety of diseases. H 2 O 2 -induced RLE-6TN cell damage is a classical model of oxidative stress injury. Studies have shown that the damage caused by oxidative stress is mediated by reactive oxygen species which plays an important role in neurodegenerative diseases [20,21]. H 2 O 2 produced in the body during metabolism is equivalent to reactive oxygen species and can aggravate the body oxidative stress injury [22,23]. The results of this study showed that H 2 O 2 induced oxidative damage and apoptosis in RLE-6TN cells which was reduced by 4,7-didehydro-neophysalin B treatment as shown in Figure 2(c). This study demonstrated that When the cellular level of ROS exceeds the body's antioxidant capacity, it induces oxidative stress leaving the cells in a redox state by producing peroxides and free radicals [24][25][26]. Mitochondrial dysfunction and DNA damage are caused by the accumulation of reactive oxygen species which mediate and accelerate apoptosis [27][28][29][30]. DNA damage causes activation of p53which serves as a major mediator of cellular stress [31]. Our results (Figure 7) indicated p53mediated apoptosis in H 2 O 2 -induced lung injury. p53 along with the members of Bcl-2 family proteins regulates proteinprotein interactions and causes activation of Bax which promotes mitochondrial membrane permeability and hence induces apoptosis [32]. In this study, 4,7-didehydro-neophysalin B suppressed p53, decreased the level of proapoptotic member Bax, and increased the level of prosurvival members Bcl-2 and Bcl-xL. Moreover, the results of apoptotic index ( Figure 2) and cell viability (Figure 4) of RLE-6TN were consistent with the observed effects on Bcl-2 family proteins. These findings suggest that 4,7-didehydro-neophysalin B has a pivotal role in regulating apoptosis and inhibiting oxidative stress-induced cell death. It is also noteworthy that some studies showed that NQO1 stabilizes the tumor suppressor p53 [33]. HO-1 upregulates Bcl-2 and Bcl-xL expressions [34] which are downstream proteins of Nrf2. A possible explanation is that 4,7-didehydro-neophysalin B protects lung injury induced by H 2 O 2 via activating the Nrf2 pathway.
The transcription factor Nrf2 plays an important role in protection against oxidative damage [35,36]. Nrf2 senses the presence of oxidative stress and regulates transcription of genes encoding cytoprotective enzymes and other proteins crucial for maintaining cellular homeostasis. Under physiological conditions, the Nrf2 inhibitor, Keap-1, which is a 9 Oxidative Medicine and Cellular Longevity negative regulator of Nrf2 binds to Nrf2 and retains Nrf2 in the cytoplasm [37]. During oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, and activates ARE-dependent gene expression including transcription of target genes NQO-1 and HO-1 [38], thereby improving the antioxidant capacity in the body [39,40]. Accumulation of Nrf2 in the nucleus is a necessary condition which is closely related to the induction of cellular defense genes [41]. The increase in cell death caused by H 2 O 2 might be attributed to the insufficient ROS removal caused by the failure of Nrf2 activation. However, Figure 5 shows that 4,7-didehydro-neophysalin B activates the antioxidant pathway affecting both Nrf2 gene levels and expression of its target proteins. Therefore, this study suggested that Nrf2 activation was required for protection of the lungs by 4,7-didehydroneophysalin B from H 2 O 2 -mediated cell death.

Conclusions
In conclusion, our study demonstrated that Physalin B attenuated H 2 O 2 -induced lung injury by regulating the Nrf2/P53 signaling pathway. Physalin B with great therapeutical potential can be used as an antioxidant agent. This study provided beneficial evidences for the application of Physalin B supplementation as an alternative treatment strategy for lung injury.

Data Availability
The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.