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. 2023 Mar 17;26(6):1204–1213. doi: 10.2174/1386207325666220715090505

Penehyclidine Hydrochloride Protects Rat Cardiomyocytes from Ischemia-Reperfusion Injury by Platelet-derived Growth Factor-B

Yan Lu 1,2, Congna Zi 3, Liang Zhang 1, Boqun Cui 1, Ling Li 2, Jun Ma 1,*
PMCID: PMC10236564  PMID: 35838232

Abstract

Aims and Objective

The lack of effective treatments for myocardial ischemia-reperfusion (MI-R) injury severely restricts the effectiveness of the treatment of ischemic heart disease. In the present research, we aimed to investigate the protective effect and molecular mechanism of penehyclidine hydrochloride (PHC) on MI-R cells.

Methods

Cell viability was quantified using CCK8. Cell apoptosis was analyzed using flow cytometry. Western blot and Elisa assays were used for the detection of target proteins.

Results

PHC pretreatment attenuated the inhibition of cell viability and decreased the percentage of apoptosis induced by simulated ischemia reperfusion (SIR). Platelet-derived growth factor B (PDGF-B) and its downstream AKT pathway were activated in PHC pretreated cells. After siRNA-PDGF-B transfection, cell viability was inhibited and apoptosis was activated in PHC pretreated SIR cells, suggesting that PHC protected cells from SIR. PDGF-B knockdown also increased the levels of CK, LDH, IL-6 and TNF-α in PHC pretreated SIR cells. The effect of AKT inhibitor on H9C2 cells was consistent with that of PDGF-B knockdown.

Conclusion

PHC pretreatment can protect cardiomyocytes from the decrease of cell activity and the increase of apoptosis caused by reperfusion through up-regulating PDGF-B to activate PI3K pathway. Our study indicates that PHC is a potential drug to protect cells from reperfusion injury and PDGF-B is a potential target for preventing MI-R injury.

Keywords: Apoptosis, cell viability, inflammatory factors, myocardial ischemia-reperfusion injury, penehyclidine hydrochloride, H9C2

1. INTRODUCTION

Ischemic heart disease (IHD) is one of the most common cardiovascular diseases worldwide [1]. Myocardial ischemia-reperfusion (MI-R) can effectively reduce the overall mortality rate [1, 2]. However, the restoration of blood flow to the previously ischemic cells will aggravate the symptoms of cell damage, including myocardial cell dysfunction and cell death [2]. The mechanism of MI-R injury has not been fully elucidated. It is generally believed that critical causes for the occurrence and development of MI-R injury include free radical burst, calcium overload, mitochondrial damage, adenosine triphosphate (ATP) energy metabolism disorder, inflammation, cell autophagy, apoptosis and necrosis during reperfusion [3-5]. In short, as a major obstacle to the treatment of ischemic cardiomyopathy, MI-R injury still lacks effective prevention and treatment strategies.

Penehyclidine hydrochloride (PHC) is a novel selective anticholinergic drug with muscarinic acetylcholine receptor (mAChR) selectivity, which has a strong anticholinergic effect on both the central and peripheral nerves [6, 7]. Due to the lack of M receptor subtype selectivity, atropine can cause tachycardia and block the presynaptic membrane M2 receptor regulatory function, which PHC can be effectively avoided with long efficacy and less side effects [6, 7]. At present, the application of PHC is progressing rapidly in clinical fields, such as pre-anaesthesia medication, organophosphorus pesticide poisoning, shock, hypertension and respiratory diseases [8-11].

Several studies have shown that PHC has a cytoprotective effect. It can stabilize the membrane structure of cell membranes, lysosomes and mitochondria, reduce the release of lysosomes, inhibit the production of arachidonic acid products and the formation of shock factors, and can also reduce the permeability of capillary wall and the inflammatory exudation reaction [6, 12, 13]. In vivo experiments have confirmed that PHC can improve microcirculation by reducing blood viscosity and fibrinogen levels, increasing the deformability of red blood cells, inhibiting the synthesis of thromboxane A (TXA) and platelet aggregation [11, 14]. During septic shock and cardiopulmonary resuscitation, PHC's effect on anti-central M and N receptor can improve cardiopulmonary microcirculation disorders and protect important organs from damage [15]. However, its role and mechanism in MI-R injury remains unclear. In the present study, rat cardiomyocytes were used to simulate MI-R injury to investigate the protective effect and molecular mechanism of PHC on cardiomyocytes. Our study confirmed that PHC was a potential drug to protect cardiomyocytes from reperfusion injury and provided a new potential target for the prevention of MI-R injury.

2. MATERIALS AND METHODS

2.1. Cell Culture and Transfection

Rat cardiomyocyte H9C2 cells were incubated in DMEM with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 (negative control, NC group). Ischemic cells were maintained in the glucose-free and serum-free medium and cultured in a hypoxic incubator with 95% N2/5% CO2 gas mixture at 37°C for 6 h (SIR group). After ischemia, the cells were incubated in DMEM with 10% FBS at 37°C for another 6 h. Cells were pretreated with 0.1 μM PHC for 2 h before ischemia (SIR& PHC group).

After growing to 70-80% confluence, H9C2 cells were transfected using LipofectAmine 2000 according to the manufacturer’s protocol. The overexpression plasmid and siRNA of platelet-derived growth factor B (PDGF-B) were all purchased from Shanghai GeneChem Co., Ltd. (China). 200 nmol/L BEZ235 was used to inhibit the AKT signaling pathway.

2.2. Sequencing and Analysis

Cells of SIR and SIR&PHC group were sequenced from BGI (China) using Illumina Hiseq. GO and KEGG Pathway Enrichment of differentially expressed genes were performed using DAVID (https://david.ncifcrf.gov/) and KOBAS (http://kobas.cbi.pku.edu.cn/), respectively.

2.3. Cell-counting-kit 8 (CCK8)

Cell viability was quantified using CCK8 (Solarbio Science and Technology Ltd., China) regent as directed by the manufacturer’s protocol. After the treatment or transfection, cells were seeded into a 96-well plate. The cell viability was detected at 0 and 24 h by adding 10 μl of CCK8 regent. After incubation for 1.5 h, the optical density (OD) value was measured at 450 nm.

2.4. Flow Cytometry for Apoptosis

After the transfection or treatment, H9C2 cells were harvested and stained with phycoerythrin (PE)-annexin V and 7-amino-actinomycin (7-AAD) (BD Biosciences, Erembodegem, Belgium) at room temperature in the dark for 15 min. The apoptotic cells were then determined by flow cytometry and measured using FlowJo 7.6 software (Flow Jo, LLC.).

2.5. Western Blot Analysis

Proteins were extracted from H9C2 cells using RIPA buffer supplemented with protease inhibitors. The protein concentration was quantified by BCA method. Then, SDS-PAGE was performed to separate the protein samples. The protein was transferred into a polyvinylidene difluoride (PVDF) membrane. After blocking with fat-free milk for 40 min, the membrane was incubated with the primary and secondary antibodies successively for 1 h. Western blot was performed using anti-p-AKT (1:2000, 66444-1-Ig, Proteintech Group, USA), AKT (1:2000, 10176-2-AP, Proteintech Group, USA), p-mTOR (1:1000, ab109268, Abcam, UK), mTOR (1:1000, ab32028, Abcam, UK), Casepase3 (1:4000, ab32351, Abcam, UK), Bcl-2 (1:1000, 12789-1-AP, Proteintech Group, USA), Bax (1:5000, 50599-2-Ig, Proteintech Group, USA) and GAPDH (1:10000, 60004-1-Ig, Proteintech Group, USA). After the incubation with ECL regent, chemiluminescence was measured using an image analyzer and quantified using ImageJ software.

2.6. Elisa

The levels of creatine kinase (CK), lactic dehydrogenase (LDH) and inflammatory factors were quantified using Elisa assay as directed by the manufacturer’s protocol. The Elisa kits for CK (ab264617), LDH (ab183367), IL-6 (ab178013), IL-10 (ab185986) and TNF-α (ab181421) were all purchased from Abcam (UK).

2.7. Statistical Analysis

GraphPad Prism 8 (GraphPad Software, USA) was used for data analysis and plotting. Comparison between groups was performed using One-way ANOVA. P-value < 0.05 was considered significant. All data were calculated from three replicates.

3. RESULTS

3.1. Construction of MI-R Injury Cell Model

H9C2 cells were incubated in DMEM with 95% N2/5% CO2 for 6 h and subsequently in DMEM with 10% FBS for another 6 h to simulate MI-R injury (SIR group). Then, the cell viability was detected by CCK8 assay. As shown in Fig. (1A), the OD value of SIR cells declined significantly compared with the untreated H9C2 cells. From the micrographs of H9C2 cells, the cell morphology has changed dramatically after simulated reperfusion. SIR cells grew in clusters, and dead cells increased significantly (Fig. 1B). The levels of MI-R injury markers, CK and LDH, also increased markedly after SIR (Figs. 1C and D). Importantly, the percentage of apoptotic cells increased significantly in SIR cells compared with the untreated H9C2 cells (Figs. 1E and F). These results proved that the MI-R injury cell model was successfully constructed.

Fig. (1).

Fig. (1)

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PHC protects H9C2 cells from reperfusion injury.

(A), H9C2 cells of simulated ischemia reperfusion (SIR) group were incubated in DMEM with 95% N2/5% CO2 at 37°C for 6 h. Cells were pretreated with PHC (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 5, 10 μM) for 2 h before SIR. Cell viability was detected using CCK8 assay. (B), cell micrographs of each group were taken after the incubation in DMEM with 95% N2/5% CO2 at 37°C for 6 h. (C) and (D), the levels of CK and LDH were detected using Elisa assay. (E), Flow cytometry was performed to quantify cell apoptosis. (F), the percentage of apoptotic cells was analyzed using FlowJo software. *P<0.05 vs. NC; #P<0.05 vs. SIR.

3.2. PHC Protects H9C2 Cells from Reperfusion Injury

Cells were pretreated with PHC (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 5, 10 μM) for 2 h before SIR. From CCK8 result, when PHC≥ 0.05 μM, the OD value rose compared with untreated SIR cells, suggesting that PHC could protect H9C2 cells from reperfusion injury. In addition, cell viability was rescued significantly by 0.1 μM PHC treatment (Fig. 1A). Therefore, 0.1 μM PHC was used for subsequent experiments. Moreover, cell micrograph and the levels of MI-R injury markers further confirmed our hypothesis. After pretreatment with 0.1 μM PHC, cell aggregation and death have been alleviated markedly (Fig. 1B), and the levels of CK and LDH also declined significantly compared with the untreated SIR cells (Figs. 1C and D). Cell flow cytometry gave consistent results. The percentage of apoptotic cells declined significantly in PHC& SIR cells compared with SIR cells (Figs. 1E and F). Our data proved that PHC protected H9C2 cells from reperfusion injury.

3.3. Analysis of the Potential Targets and Pathways by which PHC Protects Cells from Reperfusion Injury

We further investigated the potential targets and pathways by which PHC protected H9C2 cells from SIR through genome sequencing. SIR& PHC and SIR group cells were sequenced and differentially expressed genes were analyzed. As shown in Fig. (2A), there were 265 down-regulated genes and 403 up-regulated genes with statistical significance in SIR &PHC cells compared with the SIR cells.

Fig. (2).

Fig. (2)

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The potential targets and pathways by which PHC protects cells from reperfusion injury were analyzed through sequencing project. (A), Cells pretreated with 0.1 μM PHC for 2 h before SIR (SIR&PHC group) were sequencing with the SIR cells as the control. Differentially expressed genes between these two group cells were screened and a volcano plot was generated. (B), KEGG Pathway Enrichment of differentially expressed genes was performed using KOBAS (http://kobas.cbi.pku.edu.cn/). (C), Differentially expressed genes in PI3K-AKT signaling pathway. The original image KEGG graph was generated from KEGG (www.kegg.jp).

According GO analysis, differentially expressed genes were mainly distributed in cytoplasm and involved in the process of angiogenesis, cell proliferation, transcription from RNA polymerase II promoter and cell differentiation, etc. KEGG pathway analysis showed that differentially expressed genes enriched in p53, HIF-1 and PI3K-AKT signaling pathways, (Fig. 2B) etc. AKT signaling pathway is a critical hub of the regulation of cell proliferation and apoptosis, and plays an important role in MI-R injury. PDGF-B, as an upstream protein of AKT signaling pathway, was up-regulated in SIR&PHC cells with statistical significance (Fig. 2C). Therefore, we explored the mechanism of PHC to play a protective role from SIR through PDGF-B and AKT signaling pathway.

3.4. PHC Activates AKT Signaling Pathway through PDGF-B after SIR

SIR&PHC cells were transfected with siRNA-PDGF-B or treated with 200 nmol/L BEZ235 (inhibitor of AKT signaling pathway) to generate PHC & PDGF-B KD or PHC&BEZ235 group, respectively. First, we detected the phosphorylation levels of AKT and mTOR using western blot. As shown in Figs. (3A-C), after SIR, the levels of p-AKT/AKT and p-mTOR/mTOR increased significantly in PHC pretreated cells. However, siRNA-PDGF-B reduced the levels of p-AKT/AKT and p-mTOR/mTOR compared with SIR &PHC cells, suggesting that PDGF-B knockdown could block the activation of PHC to AKT pathway during the MI-R injury. In addition, the treatment of 200 nmol/L BEZ235 also declined the levels of p-AKT/AKT and p-mTOR/mTOR compared with SIR&PHC cells.

Fig. (3).

Fig. (3)

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PHC promotes cell viability through PDGF-B and AKT pathway.

(A), SIR&PHC cells were transfected with siRNA-PDGF-B to generate PHC&PDGF-B KD group, and were treated with 200 nmol/L BEZ235 to generate PHC&BEZ235 group. Western blot was performed to detect the phosphorylation levels of AKT and mTOR. (B) and (C), relative levels of p-AKT/AKT and p-mTOR/mTOR were measured using ImageJ software. *P<0.05 vs. SIR; #P<0.05 vs. SIR&PHC. (D), CCK8 was used to detect cell viability. *P<0.05; ***P<0.001.

3.5. PHC Promotes Cell Viability through PDGF-B and AKT Pathway after SIR

From CCK8 results, siRNA-PDGF-B declined the OD value compared with SIR&PHC cells after SIR, suggesting that PDGF-B knockdown could block the protection of PHC against SIR (Fig. 3C). The treatment of 200 nmol/L BEZ235 also reduced cell viability compared with SIR&PHC group, indicating that the inhibition of AKT also blocked the protection of PHC to H9C2 cells during the MI-R injury (Fig. 3C). Therefore, we hypothesized that PHC protected cardiomyocytes from reperfusion injury through upregulating PDGF-B and activating AKT pathway.

3.6. PHC Inhibits Cell Apoptosis through PDGF-B and AKT Pathway

To further verify our hypothesis, cell apoptosis was quantified using flow cytometry. As shown in Fig. (4A and B), siRNA-PDGF-B (12.27 ± 0.59%) increased the percentage of apoptotic cells significantly compared with the SIR&PHC cells (9.53 ± 0.90%). AKT inhibitor (25.74 ± 0.85%) also increased the percentage of apoptotic cells significantly compared with the SIR&PHC cells (9.53 ± 0.90%) (Figs. 4A and B).

Fig. (4).

Fig. (4)

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PHC inhibits cell apoptosis through PDGF-B and AKT pathway.

(A), Flow cytometry was performed to quantify cell apoptosis. (B), the percentage of apoptosis cells was analyzed using FlowJo software. (C), the expression levels of apoptosis-related proteins were detected using western blot. The relative levels of Bcl-2 (D), Bax (E), Casepase3-p17 (F) and Caspase3 (G) were quantified using ImageJ software. *P<0.05 vs. SIR; #P<0.05 vs. SIR&PHC.

The expression of apoptotic factors, Bax and Casepase3 -p17, increased after the transfection of siRNA-PDGF-B. On the contrary, the expression of Bcl-2 and Total-Caspase3 declined after the transfection of siRNA-PDGF-B (Figs. 4C-G). In addition, AKT inhibitor promoted the expression of Bax and Casepase3-p17, while inhibiting the expression of Bcl-2 and Total-Caspase3 compared with the SIR&PHC cells (Figs. 4C-G). These results proved that PHC inhibited cell apoptosis through PDGF-B and AKT pathway after SIR.

3.7. PHC Inhibits the Expression of Inflammatory Factors through PDGF-B and AKT Pathway

Finally, we detected the levels of inflammatory factors using the Elisa assay. As shown in Figs. (5A and B), the transfection of siRNA-PDGF-B increased the levels of MI-R injury markers, CK and LDH, compared with the SIR&PHC cells. AKT inhibitor also increased the levels of CK and LDH. Importantly, inflammatory factors, IL-6 and TNF-α, were up-regulated by siRNA-PDGF-B and AKT inhibitor after SIR in PHC pretreated cells, while IL-10 was down-regulated by siRNA-PDGF-B and AKT inhibitor (Figs. 5C-E). These data indicated that PDGF-B knockdown or AKT inhibitor blocked the protection of PHC to SIR in H9C2 cells.

Fig. (5).

Fig. (5)

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PHC inhibits the expression levels of inflammatory factors through PDGF-B and AKT pathway.

Elisa assay was performed to detect the levels of inflammatory factors. The relative levels of CK (A), LDH (B), IL-6 (C), IL-10 (D) and TNF-α (E) were analyzed. *P<0.05 vs. SIR; #P<0.05 vs. SIR&PHC.

4. DISCUSSION

In the present research, we investigated the protective effect of PHC on MI-R injury cells in an in vitro cell model. H9C2 cells were incubated in glucose- and serum-free medium for 6 h under hypoxia and then in DMEM with 10% FBS for another 6 h to construct MI-R injury cell models in rat cardiomyocytes. In SIR group, the percentage of apoptosis and the activities of LDH and CK were significantly increased, while cell viability declined, indicating that the model was successfully constructed. Ischemia and hypoxia can induce the high expression of apoptosis genes Bcl-2/Bax, p53, BCL XL, Fas/Fas-L and tumor necrosis factor in the nucleus, start the apoptosis process and aggravate the apoptosis process [16, 17]. After ischemia reperfusion, although the blood supply is restored, the production of reactive oxygen species (ROS) and the overload of calcium ions will initiate the apoptosis process of myocardial cells [18, 19]. Studies have shown that apoptosis plays an important role in the damage of myocardial structure and function in the process of MI-R injury, and is the target of specific drugs to intervene MI-R injury [20, 21]. Our studies showed that PHC could inhibit the apoptosis induced by MI-R injury in rat cardiomyocytes, indicating that PHC has a certain protective effect on cell injury, and it is a potential drug to prevent MI-R injury.

Previous studies have shown that PHC can significantly improve microcirculation and compensatory cardiac function and shorten the time of shock resuscitation, thus reducing the injury of visceral organs during reperfusion aftershock [15, 22]. Studies have suggested that PHC can alleviate the ischemia-reperfusion injury of skeletal muscle and stomach induced by limb ischemia-reperfusion, which may be due to the inhibition of inflammatory cytokines and active oxygen, and the alleviation of calcium overload after PHC intervention [23]. However, the specific mechanism of PHC protection against ischemia-reperfusion injury remains unclear. Therefore, we analyzed the difference at transcriptome level between PHC treated and untreated MI-R injury cells through sequencing. PDGF-B was found to be significantly upregulated by PHC in H9C2 cells.

As a major mitogenic agent in vivo, PDGF can stimulate the proliferation, migration and chemotaxis of various tissues and cells [24]. It is involved in human embryonic development, normal physiological activities, disease occurrence and tissue repair after various injuries [25-27]. PDGF was originally isolated from platelets and named after it. Subsequently, it was found that mesenchymal cells such as macrophages, fibroblasts, endothelial cells, vascular smooth muscle cells, epidermal cells and other mesenchymal cells could secrete PDGF [28]. PDGF-B encodes one of the four peptide chains of PDGF (PDGF-A, -B, -C and -D) [28]. Research shows that PDGF-B signaling protects rat cardiac allografts from ischemia-reperfusion injury (IRI) after heart transplantation by promoting endothelial and cardiomyocyte recovery [29]. We found that the protective effect of PHC on cell injury was blocked by PDGF-B knockdown in PHC treated cells, which confirmed that PHC protected cells from reperfusion injury by activating PDGF-B.

We further analyzed the downstream pathway of PHC in SIR cells through PDGF-B. According to the sequencing results, the PI3K signaling pathway was abnormally activated by PHC treatment. In the cell model, AKT inhibitor significantly blocked the inhibition of PHC on apoptosis, suggesting that PHC prevented reperfusion injury by activating PI3K signaling pathway. Studies have confirmed that PI3K translocated from the cytoplasm to the cytoplasmic membrane, binds to phosphorylated PDGF through the sH2 domain, and then active Ras binds to the catalytic subunit of PI3K to fully activate PI3K. The activated PI3K can then activate multiple downstream pathways such as protein kinase c (PKc) and protein kinase B (PKB) [30]. The activation of PI3K is essential for PDGF-B to regulate cell proliferation, migration, cell attachment and apoptosis [25]. In addition, AKT inhibitors are more effective in blocking the protective effect of PHC than PDGF-B knockout, which may be due to the presence of other targets or potential feedback regulation to rescue the activation of AKT pathway.

Previous studies have shown that PHC pretreatment attenuated apoptosis by ameliorating the imbalance of IRI mitochondrial dynamics, while significantly improving cardiac function by reducing infarct size and myocardial enzyme level, thereby playing a long-term cardioprotective role in the rat model of MI / R injury [31]. PHC can also significantly reduce the levels of TNF-α, IL-1β, IL-6, prostaglandin E2 (PGE2) and myocardial cyclooxygenase 2 (COX-2) in MI/R-injured rats [32]. PHC precondition also activated p38MAPK and JNK signaling pathways. p38MAPK inhibitor (SB239063) and JNK inhibitor (SP600125) have the same effect as PHC, ameliorating myocardial abnormalities, mitochondrial abnormalities and excessive oxidative stress in MI/R-injured rats [33]. The up-regulation of PDGF-B to activate PI3K pathway may be connected to the regulation of cardiac mitochondrial dynamics. In another study, PHC post-treatment 5 to 10 min after reperfusion down-regulated the expression of NF-κB and inflammatory factors, and significantly up-regulated IκB-α expression [34]. Importantly, there is crosstalk between the activation of NF-κB and the transcription of PDGF-B. Therefore, PHC-mediated regulation of PDGF-B and AKT pathways may involve more interference mechanisms, which need to be further investigated.

CONCLUSION

In conclusion, PHC pretreatment can protect cardiomyocytes from apoptosis caused by reperfusion through up-regulating PDGF-B to activate PI3K signaling pathway, suggesting that PHC is a potential drug to protect cells from reperfusion injury.

ACKNOWLEDGEMENTS

Declared none.

LIST OF ABBREVIATIONS

MI-R

Myocardial ischemia-reperfusion

PHC

Penehyclidine hydrochloride

SIR

Simulated ischemia reperfusion

PDGF-B

Platelet-derived growth factor B

IHD

Ischemic heart disease

ATP

Adenosine triphosphate

mAChR

Muscarinic acetylcholine receptor

TXA

Thromboxane A

FBS

Fetal bovine serum

OD

Optical density

PE

Phycoerythrin

7-AAD

7-amino-actinomycin

PVDF

Polyvinylidene difluoride

CK

Creatine kinase

LDH

Lactic dehydrogenase

ROS

Reactive oxygen species

IRI

Ischemia-reperfusion injury

PKc

Protein kinase c

PKB

Protein kinase B

PGE2

Prostaglandin E2

COX-2

Myocardial cyclooxygenase 2

AUTHORS’ CONTRIBUTIONS

YL and LW mainly performed the experiments; YL analyzed the data and wrote the paper. LZ, BC and LL helped with the experiments. JM helped modify the paper. All authors had edited and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study has been conducted on Rat Myocardial Cell lines therefore no ethical approval is required.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The authors confirm that the data supporting the findings of this study are available within the article.

FUNDING

This study was supported by the National Natural Science Foundation of China [grant numbers: 81471902 and 81871592], the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support [grant number: ZYLX201810].

CONFLICT OF INTEREST

The authors declare no conflict of interest financial or otherwise.

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Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

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