PRAT 1 Cistanches Deserticola PhG-RE Through Inhibiting ERS Apoptosis Mechanism To Protect Myocardial Cell Apoptosis From H2O2- Induced Endoplasmic Reticulum Stress

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Department of Pharmacy, China-Japan Union Hospital of Jilin University, Changchun 130033, China

Correspondence should be addressed to Qian Yu; yuqian@jlu.edu.cn

Received 9 June 2020; Revised 15 July 2020; Accepted 24 July 2020; Published 25 September 2020

Academic Editor: Michel Mansur Machado

Copyright © 2020 Tianwei Lan and Qian Yu. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited.

The herb Cistanche deserticola has some myocardial protective effects. This study attempted to explain the mechanism by which PhG-RE protects myocardial cells and verify if this protection occurs through regulating the apoptosis mechanism associated with endoplasmic reticulum stress (ERS). Rat myocardial cells were exposed to 150 μg mL−1 PhG-RE for 24 h and then to 100 μmol mL−1 H2O2 for 18 h to induce ERS and establish a cell damage model. Thapsigargin (TG), a specific ERS activator, and 4- phenyl butyric acid (4-PBA), an ERS inhibitor, were used to validate the accuracy of the experiment. Our results demonstrated that PhG-RE significantly improved cell viability, protected cells, and reduced cell damage and apoptosis. PhG-RE played a role similar to that of the ERS inhibitor 4-PBA in protecting myocardial cells against apoptosis and damage induced by ER stress. Furthermore, PhG-RE significantly attenuated the mRNA expression of the ERS-associated apoptotic factors GRP78, CHOP, and Caspase-12 and the protein expression of the ERS-associated apoptotic factors GRP78, CHOP, Caspase-12, and p-JNK. Taken together, these findings suggest that PhG-RE can effectively protect myocardial cells and reduce cell apoptosis and damage, which may be related to the regulation of ERS-associated apoptosis.

1. Introduction

Cistanche deserticola (C. deserticola) is one of the herbal plants used in traditional Chinese medicine that grows in desert environments. The phenylethanoid glycoside-rich extract (PhG-RE) is the main active component of C. deserticola. Research has found that PhG-RE can protect the myocardium against ischemia-reperfusion (I/R) injury [1].

When ERS starts to occur in myocardial cells, the level of glucose-regulated protein 78 (GRP78) increases to antagonise an ERS-induced injury. As ERS progresses, apoptosis is initiated, and the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), c-Jun N-terminal kinase (JNK), and cysteinyl aspartate specific proteinase-12 (Caspase-12) increases. For example, the expression of GRP78, CHOP, and cleaved ATF6 (c-ATF6) in myocardial cells of rats with HF is significantly increased, and PERK (p- PERK), IRE1 (p-IRE1), and p-JNK are activated [2]. In addition, ERS induced by I/R injury increases the expression of GRP78 and CHOP [3]. The phosphorylation levels of PERK and eIF2α are increased [4]. The expression of pro-apoptotic proteins Bax and Caspase-3 are increased, causing cell rupture and myocardial remodeling [5], and the area of myocardial infarction and myocardial enzymatic activity is increased [3]. It was found during our previous research that PhG-RE can protect the myocardium against ischemia-reperfusion injury and can significantly reduce the area of myocardial infarction induced by I/R [1]. However, it is unclear whether PhG-RE can inhibit ERS-associated apoptosis and thereby reduce myocardial cell loss and apoptosis. In this study, cells were pretreated with PhG-RE to observe whether PhG-RE can inhibit H2O2-induced ERS and therefore reduce myocardial cell loss and apoptosis. Thapsigargin (TG), a specific ERS activator, and 4-phenyl- butyric acid (4-PBA), an ERS inhibitor, were used to further verify whether mediating ERS can effectively reduce cell apoptosis and whether this process is related to the expression levels of GRP78, CHOP, JNK, and Caspase-12, which have been shown to mediate ERS-associated apoptosis.

Cistanche can prevent cell apoptosis

2. Materials and Methods

2.1. Reagents.

The materials and methods section should be exhaustive so that all procedures can be repeated. It may be divided into headed subsections if several methods are described. H9c2 rat myocardial cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). C. deserticola Cistanche deserticola PhG-RE was obtained from Changchun Medicinal Material Co. (Changchun, China), identified by Dr. Zhong-ying Liu (School of Pharmaceutical Sciences, Jilin University) and deposited in the herbarium of the Department of Pharmacy, Jilin University (Voucher specimen number: YQCD2014). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from GIBCO (Grand Island, NY). Thapsigargin and 4-PBA were obtained from Sigma (St. Louis, MO). A lactate de- hydrogenase (LDH) activity assay kit and CCK-8 kit were purchased from Dojindo Molecular Technologies (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from HyClone (Utah, USA). An Annexin V-FITC cell apoptosis assay kit was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin was purchased from Beijing Solarbio Life Sciences (Beijing, China). A ReverTra Ace qPCR RT kit was purchased from TOYOBO (Tokyo, Japan). FastStart Universal SYBR Green master mix (Rox) was purchased from Roche (Basel, Switzerland).

2.2. Antibodies.

Rabbit polyclonal GADD153/CHOP and rabbit polyclonal GRP78/HSPA5 Abs were purchased from Novus (Colorado, USA). The mouse/rat caspase-12 affinity purified polyclonal Ab was purchased from R&D Systems (Minnesota, USA). Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) rabbit and β-actin (13E5) rabbit mAbs were purchased from Cell Signalling Technology (Massachusetts, USA). Fluorescent secondary Abs were purchased from LI-COR Biosciences (Nebraska, USA).

2.3. Groupings and Treatment.

H9c2 cells in the logarithmic growth phase were selected and treated in the following groups: (1) control group: H9c2 myocardial cells were cultured in a complete medium; (2) PhG-RE group: cells were treated for 24 h in a medium containing 150 μg mL−1 PhG-RE; (3) ERS-induced injury model group (H2O2 group): cells were treated for 18 h in a medium containing 100 μmol mL−1 H2O2; (4) (PhG-RE) –H2O2 group: cells were treated for 24 h in a medium containing 150 μg mL−1 PhG- RE and then for 18 h in 100 μmol mL−1 H2O2; (5) (4-PBA)

–H2O2 group: cells were treated for 24 h in a culture medium containing 5 mmol·mL−1 4-PBA and then for 18 h in 100 μmol·mL−1 H2O2; (6) TG group: cells were treated for

18 h in a medium containing 50 nmol·mL−1 TG; and (7) (PhG-RE) −TG: cells were treated for 24 h in a medium containing 150 μg·mL−1 PhG-RE and then for 18 h in 50 nmol·mL−1 TG.

2.4. Cell Culture.

H9c2 myocardial cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C in an incubator with 5% CO2. The complete medium was replaced every 2 d. The cells were observed under a microscope, and they grew well in a fusiform shape. When the growth density reached 70%–85%, the cells were sub-cultured at a 1: 4 ratio.

2.5. CCK-8 Assessment of Cell Viability.

H9c2 cells were microscopically examined, and the cells in the logarithmic growth phase were selected. The cell suspension was obtained through a trypsin digestion and centrifuged for 5 min in a 5 mL centrifuge tube at 1000 r min−1. After the residual liquid was discarded, 3 ml of DMEM complete medium was used to resuspend the cell pellets in 100 μL per well, and

5.0 103 cells were counted under the microscope. The cells were transferred to a 96-well plate and cultured in an incubator (37°C, 5% CO2) for 24 h to obtain completely adherent cells. The optical density (OD) was measured at a wavelength of 450 nm.

Cell viability (%) (OD experimental group − OD blank group)/(OD control group − OD blank group) × 100%.

2.6. Spectrophotometric Measurement of Cell Injury.

Lactate dehydrogenase (LDH) is an enzyme in the cytoplasm that is released upon cell injury, which then stably exists in the culture medium. The degree of cell injury can be determined by measuring the activity of LDH in the medium. This experiment was conducted according to the instructions for the kit. The OD was measured at a wavelength of 490 nm. There were three wells in each group.

2.7. Apoptosis Detection with Annexin V-FITC/PI Staining.

The supernatant was removed and transferred to a flow cytometry tube. A cell suspension was prepared with adherent cells at the bottom of the trypsin digestion vessel. After the suspension was centrifuged for 6 min at 1.2 103 r min−1, the supernatant was discarded, and the cell pellets were washed thrice with PBS. This experiment was conducted according to the instructions for the Annexin V-FITC cell apoptosis kit. After the cells were cultured for 15 min at room temperature in the dark, the cells were analyzed with a flow cytometer.

2.8. RT-qPCR Measurement of the Relative mRNA Expression of GRP78, CHOP, JNK, and Caspase-12.

Total cellular RNA was extracted with a TRIzol reagent. Two microlitres of total RNA were separated by gel electrophoresis to evaluate the extraction integrity. A spectrophotometer was used to measure and calculate the OD (260)/OD (280) value. Total RNA was reverse transcribed to produce cDNA according to the instructions for the TOYOBO ReverTra Ace qPCR RT kit. Table 1 shows the list of primers. According to the instructions for the Roche FastStart Universal SYBR Green master mix (Rox), the reaction conditions were as follows: initial denaturation at 95°C for 10 min, 95°C for 10 s, 58°C for 30 s, and 72°C for 30 s for a total of 45 cycles.

2.9.Western Blot Analysis of the Protein Expression of GRP78, CHOP, JNK, p-JNK, and Caspase-12.

Protein lysate (50 μg) was separated by electrophoresis on a 10% separating gel, followed by transfer to a PVDF membrane. The PVDF membrane was blocked in the TBS-Resolution containing 5% skimmed milk at room temperature for 90 min. The blocked PVDF membrane was incubated overnight with antibodies 1, 2, 3, and 4 at 4°C, and then for 90 min with goat anti-rabbit IgG (1: 1000) at room temperature. Finally, the membrane was developed with a two-color infrared laser imaging system.

2.10. Statistical and Analytical Methods.

The data in this study were processed with SPSS 24.0 software, and the results are presented as the means ± SD. The data from multiple groups were compared with ANOVA, while the data from two groups were compared with t-tests. A value of P < 0.05 was considered to have statistical significance. ImageJ software was used to quantitatively analyze the greyscale values of the protein bands.

Phenylethanoid Glycosides in cistanche

3. Results

3.1. Influence of PhG-RE on Cell Viability.

As shown in Figure 1, compared to that of the control group, the viability of H9c2 cells in the PhG-RE group did not change significantly (P > 0.05), but the viability of cells in both the H2O2 and TG groups decreased significantly. Compared to that of the H2O2 group, the viability of cells in both the (PhG-RE)- H2O2 and the (4-PBA)-H2O2 groups increased significantly. Compared to that of the TG group, cell viability in the (PhG- RE)-TG group increased significantly. All of the results were statistically significant (P < 0.01).

3.2. Influence of PhG-REon Cell Injury.

As shown in Figure 2, although the cells in the PhG-RE group were damaged compared to those in the control group, that damage did not reach statistical significance (P > 0.05). Cell injury in the H2O2 and TG groups increased significantly. Compared to that of the H2O2 group, cell injury in the (PhG-RE)-H2O2 and (4-PBA)-H2O2 groups decreased significantly (P < 0.01). Compared to that of the TG group, cell injury in the (PhG-RE)-TG group decreased significantly (P < 0.01).

3.3. Influence of PhG-RE on Cell Apoptosis.

As shown in Figure 3, compared to that of the control group, cell apoptosis in the PhG-RE group did not change significantly (P > 0.05), while that in the H2O2 and TG groups increased significantly (P < 0.01). Compared to that of the H2O2 group, cell apoptosis in the (PhG-RE)-H2O2 and (4-PBA)-H2O2

groups decreased significantly (P < 0.01). Compared to that of the TG group, cell apoptosis in the (PhG-RE)-TG group decreased significantly (P < 0.01).

3.4. Influence of PhG-RE on the Expression of GRP78, CHOP, JNK, and Caspase-12 mRNA.

The relative mRNA expression in each experimental group was quantitatively measured with RT-qPCR. As shown in Figure 4, compared to that of the control group, the relative mRNA expression of GRP78, CHOP, and Caspase-12 increased significantly (P < 0.01) in the H2O2 group, and the relative mRNA expression of JNK also increased (P < 0.05). In the TG group, the relative mRNA expression of GRP78, CHOP, JNK, and Caspase-12 increased significantly (P < 0.01). Compared to that of the H2O2 group, the relative mRNA expression of GRP78, CHOP, and Caspase-12 in the (PhG-RE)-H2O2 and (4- PBA)-H2O2 groups decreased significantly (P < 0.01), while the relative mRNA expression of JNK did not change sig- nificantly (P > 0.05). Compared to that of the TG group, the relative mRNA expression of GRP78, JNK, and Caspase-12 in the (PhG-RE)-TG group decreased (P < 0.01), and the relative mRNA expression of CHOP did not change sig- nificantly (P > 0.05).

3.5. Influence of PhG-RE on the Protein Expression of GRP78, CHOP, and Caspase-12.

Figure 5 shows the protein expression levels of GRP78, CHOP, and Caspase-12. Compared to that of the control group, the expression of these three proteins in the H2O2 and TG groups increased sig- nificantly after the cells were treated (P < 0.01). Compared to that of the H2O2 group, the expression of GRP78, CHOP, and Caspase-12 decreased significantly in the (PhG-RE)- H2O2 and (4-PBA)-H2O2 groups (P < 0.01). Compared to that of the TG group, the expression of GRP78, CHOP, and Caspase-12 in the (PhG-RE)-TG group decreased significantly.

3.6. Influence of PhG-RE on JNK and p-JNK.

As shown in Figure 6, compared to that of the control group, the expression of p-JNK in the H2O2 and TG groups increased significantly (P < 0.01). Compared to that of the H2O2 group, the expression of p-JNK in the (PhG-RE)-H2O2 and (4- PBA)-H2O2 groups decreased significantly (P < 0.01). Compared to that of the TG group, the expression of p-JNK in the (PhG-RE)-TG group decreased significantly (P < 0.01). Concerning the expression of JNK, there were no significant changes in any of the experimental groups (P > 0.05).

Cistanche can prevent cell apoptosis