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Effect of PHC on cardiomyocyte apoptosis. (A) Representative images of flow cytometry. Cell populations were regarded, respectively, as early‑stage apoptotic in the LR quadrant, living in the LL quadrant, late‑stage apoptotic in the UR quadrant and necrotic in the UL quadrant. (B) Representative images of myocardial TUNEL staining. TUNEL‑positive cells were regarded as apoptotic and counted using a microscope at x400 magnification. Cells in the nucleus contained brown granules suggesting positive staining and are indicated by red arrows. Normal cell nuclei were stained blue with hematoxylin. (C) The cardiomyocyte apoptotic rate was expressed as a ratio of TUNEL‑positive cells (apoptotic cells) to the total number of cardiomyocytes. Data were expressed as the mean ± standard deviation (n=6 each group). Scale bar, 20 µm. ## P<0.01 vs. sham; ** P<0.01 vs. I/R. ns, not significant (P>0.05); I/R, ischemia/reperfu‑ sion; PHC, penehyclidine hydrochloride; TUNEL, terminal deoxynucleotidyltransferase‑mediated dUTP nick end labelling; LL, lower left; UL, upper left; LR, lower right; UR, upper right; FITC, fluorescein isothiocyanate.
Source publication
Ischemic heart disease is a major cause of mortality and disability worldwide. Timely reperfusion is currently the most effective method of treating ischemic heart disease; however, abrupt reperfusion may cause ischemia/reperfusion (I/R) injury. Apoptosis serves an important role in the progression of myocardial I/R injury and it has been demonstra...
Contexts in source publication
Context 1
... of PHC on cardiomyocyte apoptosis. Following 3 h reperfusion, apoptotic cells were analyzed using flow cytom- etry ( Fig. 2A). There was a decreased proportion of apoptotic cells in the PHC+sham group compared with the Sham group; however, this difference was not significant (5.52±2.32% vs. 6.20±2.93%). The proportion of apoptotic cells in the I/R group was significantly increased compared with the Sham group (25.12±5.92% vs. 6.20±2.93%) and significantly ...
Context 2
... Sham group; however, this difference was not significant (5.52±2.32% vs. 6.20±2.93%). The proportion of apoptotic cells in the I/R group was significantly increased compared with the Sham group (25.12±5.92% vs. 6.20±2.93%) and significantly decreased in the PHC+I/R group compared with the I/R group (7.35±3.42% vs. 25.12±5.92%, both P<0.001; all Fig. ...
Context 3
... 3 h reperfusion, TUNEL staining was perfo- rmed ( Fig. 2B) to measure the rate of apoptosis as the proportion of apoptotic cells in the sample. The difference in the apoptotic rate of cardiomyocytes in the PHC+sham group compared with the Sham group was not significant (3.62±1.5% vs. 2.95±1.4%; Fig. 2C). The apoptotic rate was significantly increased in the I/R group compared with the Sham ...
Context 4
... 3 h reperfusion, TUNEL staining was perfo- rmed ( Fig. 2B) to measure the rate of apoptosis as the proportion of apoptotic cells in the sample. The difference in the apoptotic rate of cardiomyocytes in the PHC+sham group compared with the Sham group was not significant (3.62±1.5% vs. 2.95±1.4%; Fig. 2C). The apoptotic rate was significantly increased in the I/R group compared with the Sham group (58.09±6.2% vs. 2.95±1.4%) and significantly decreased in the PHC+I/R group compared with the I/R group (15.84±2.6% vs. 58.09±6.2%, both P<0.01; Fig. ...
Context 5
... in the PHC+sham group compared with the Sham group was not significant (3.62±1.5% vs. 2.95±1.4%; Fig. 2C). The apoptotic rate was significantly increased in the I/R group compared with the Sham group (58.09±6.2% vs. 2.95±1.4%) and significantly decreased in the PHC+I/R group compared with the I/R group (15.84±2.6% vs. 58.09±6.2%, both P<0.01; Fig. ...
Citations
... Based on this study and other PHC and MIRI studies, it is clear that PHC inhibits MIRI and can be used as a candidate drug to treat MIRI. This study revealed the inhibitory effect of PHC pretreatment on MIRI, and previous studies revealed that PHC pre/posttreatment alleviated MIRI through different pathways [23,38]. This proved the consistency of PHC treatment on MIRI inhibition. ...
Penehyclidine hydrochloride (PHC) is an anticholinergic drug with cardioprotective effects. Ferroptosis is closely related to myocardial ischaemia-reperfusion injury (MIRI). In the present study, MIRI was induced in rats by left anterior descending coronary artery ligation. PHC pretreatment increased haemodynamic parameters and histopathological damage and reduced myocardial infarction size in the MIRI model. PHC pretreatment also inhibited ferroptosis, which was characterized by the decreased levels of Fe²⁺, 4-hydroxynonenal and ACSL4, and increased levels of GPX4, GSH-Px and GST. In response to 6 h of oxygen-glucose deprivation and 18 h of reoxygenation, PHC pretreatment had the same effects on these factors in H9c2 cells and reduced lipid ROS levels. Furthermore, ACSL4 overexpression reversed the protective effects of PHC on H9c2 cells. These results indicated that PHC inhibited MIRI through ACSL4-mediated ferroptosis. This study demonstrated that PHC could inhibit ferroptosis in MIRI and the relationship among PHC, ACSL4, ferroptosis and MIRI.
Graphical Abstract
This study demonstrated the inhibitory effect of PHC on ferroptosis and showed that PHC affects MIRI through ACSL4-mediated ferroptosis in vivo and in vitro.
... Studies have demonstrated that VDAC1 oligomerization induces the formation of large channels that facilitate the passage of folded proteins, such as CYCS (Hosaka et al., 2017;. Continued opening of the large channels due to VDAC1 oligomerization also decreases the membrane potential (Jiao et al., 2022;Tan et al., 2017). In addition, we found that VDAC1 oligomerization may be involved in the process of necroptosis, which was not previously reported. ...
Ischemia-reperfusion (I/R) injury is a common pathological mechanism in many retinal diseases, which can lead to cell death via mitochondrial dysfunction. Voltage-dependent anion channel 1 (VDAC1), which is mainly located in the outer mitochondrial membrane, is the gatekeeper of mitochondria. The permeability of mitochondrial membrane can be regulated by controlling the oligomerization of VDAC1. However, the functional mechanism of VDAC1 in retinal I/R injury was unclear. Our results demonstrate that oxygen-glucose deprivation and re-oxygenation (OGD/R) injury leads to apoptosis, necroptosis, and mitochondrial dysfunction of R28 cells. The OGD/R injury increases the levels of VDAC1 oligomerization. Inhibition of VDAC1 oligomerization by VBIT-12 rescued mitochondrial dysfunction by OGD/R and also reduced apoptosis/necroptosis of R28 cells. In vivo, the use of VBIT-12 significantly reduced aHIOP-induced neuronal death (apoptosis/necroptosis) in the rat retina. Our findings indicate that VDAC1 oligomers may open and enlarge mitochondrial membrane pores during OGD/R injury, leading to the release of death-related factors in mitochondria, resulting in apoptosis and necroptosis. This study provides a potential therapeutic strategy against ocular diseases caused by I/R injury.
... 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]. ...
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.
Method
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.
Result
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 the 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.
... The MDA level was also decreased in OGD/R-stimulated HT-22 cells transfected with Ad-shPKM2, revealing the protective effect of PKM2 knockdown against OGD/R-induced cell damage. Furthermore, since apoptosis [31] and oxidative stress (reactive oxygen species) [32] play important role in the progression of cerebral I/R injury, the effects of PKM2 on cell apoptosis and reactive oxygen species accumulation in OGD/R-stimulated HT-22 cells need further investigation. + + + + shNC + ----shPKM2 + + ---HMGB1 + ---- In addition to cell damages, inflammatory response also serves as dominating pathogenic factor for ischemic stroke [33], which is associated with all the phases of cerebral I/R injury [34]. ...
Objectives:
Cerebral ischemia-reperfusion (I/R) injury is the leading cause of ischemic stroke. Pyruvate Kinase isozymes M2 (PKM2), as a critical glycolytic enzyme during glycolysis, is involved in neuronal apoptosis in rats with hypoxic-ischemic encephalopathy. This study focused on functional investigation and potential molecular mechanism toward PKM2 in cerebral I/R injury.
Methods:
Cerebral I/R injury model was established by middle cerebral artery occlusion (MCAO) in vivo or oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. qRT-PCR and Western blot were used to detect the expression of PKM2 in I/R injury models. The effects of PKM2 on I/R injury were determined via triphenyl tetrazolium chloride staining and evaluation of neurological deficits. Cell Counting Kit-8 was employed to detect cell viability, and ELISA was conducted to detect pro-inflammatory cytokines. The underlying mechanism involved in regulation of PKM2 on I/R injury was investigated via ELISA and Western blot.
Results:
PKM2 was upregulated after cerebral I/R injury. Knockdown of PKM2 alleviated MCAO-induced infarction and neurological dysfunction. Moreover, PKM2 knockdown also alleviated OGD/R-induced neuronal cell injury and inflammatory response. Mechanistically, PKM2 knockdown-induced neuroprotection was accompanied by inhibition of high-mobility group box 1 (HMGB1), reflected by inactivation of TLR4/MyD88 (myeloid differentiation factor 88)/TRAF6 (TNF receptor-associated factor 6) signaling pathway.
Conclusions:
Knockdown of PKM2 attenuated cerebral I/R injury through HMGB1-mediated TLR4/MyD88/TRAF6 expression change, providing a potential target for cerebral I/R injury treatment.
... These results, together, were consistent with the pathological evaluation of myocardial tissues. Ischemia triggers apoptosis of cardiomyocytes and then expands and partially contributes to cardiac death through reperfusion (27). Song et al. reported that I/R injury markedly induced activation of 12/15-LOX and resulted in cardiomyocyte apoptosis, increased caspase-3 activity, and Bax/Bcl-2 ratio (28). ...
Background:
Myocardial ischemia/reperfusion (I/R) injury has become a global public health concern. An increasing amount of evidence has shown that polyphyllin I (PPI) has anti-apoptotic and antioxidant functions. This study was performed to evaluate the cardioprotective effects of PPI in a rat model of myocardial I/R injury and the underlying mechanism.
Methods:
We exposed induced a rat model of I/R injury by exposing rat hearts to left anterior descending coronary artery ligation for 30 min, followed by 24 h of reperfusion. Cardiac function was analyzed by echocardiography and HE staining. Myocardial apoptosis, inflammation, and oxidative stress were detected to analyze the PPI's role in I/R injury.
Results:
The results showed that pretreatment with PPI improved impaired histological morphology, as shown by histopathological examination. Echocardiography analysis showed that PPI increased the levels of HR, left ventricular ejection fraction (LVEF), and left ventricular wall thickness (LVWT), accompanied by decreased left ventricular end-systolic volume (LVESV). Also, PPI decreased the expression of CK-MB, Mb, cTnI, and LDH. Specifically, PPI also changed the expression of apoptotic makers (Caspase-3, Bax, and Bcl-2), inflammatory cytokines (TNF-α, IL-6, iNOS, and IL-10) and oxidative stress markers (SOD, GSH, ROS, and MDA). Notably, western blot (WB) showed that PPI treatment inhibited the phosphorylation activity of NF-κB p65.
Conclusions:
The findings showed that PPI exerted a favorable protective effect on I/R injury by inhibiting the inflammatory response and oxidative stress. It offered new drug candidates for the treatment of myocardial I/R injury.
... Similarly, Han et al. [31] reported that I/R-induced myocardial injury caused an increase in caspase-3 expression in cardiac myofibrils. In addition, Tan et al. [32] reported an increase in the TUNEL and caspase-3 expression following I/R. We also found an increase in the caspase-3 expression in cardiac myofibrils. ...
Background:
The aim of this study was to investigate the potential protective effect of whortleberry by examining the effects on heart tissue at the molecular level of ischemia-reperfusion injury caused by surgical repair of a ruptured abdominal aortic aneurysm.
Methods:
Between May 2018 and February 2019, a total of 32 male Sprague-Dawley rats were randomly assigned into control, sham (ischemia-reperfusion+glycerol), ischemia-reperfusion, and ischemia-reperfusion+whortleberry groups. Hypovolemic shock was applied to the rats in the ischemia-reperfusion groups for one hour. The abdominal aorta was explored following midline laparotomy and atraumatic microvascular clamps were applied from the infrarenal level. Following one-hour ischemia, the clamps were removed, and reperfusion was established for two hours. In the sham group, intraperitoneal glycerol once daily was applied five days before surgery. In the whortleberry group, whortleberry treatment was administered via the intraperitoneal route five days before ischemia-reperfusion.
Results:
The ischemia-reperfusion group exhibited a decrease in the glutathione levels and an increase in the malondialdehyde levels (p<0.01 and p<0.01, respectively). We also observed an increase in the caspase-3 positivity in cardiac myofibrils (p<0.01). Whortleberry administration lowered both malondialdehyde levels and numerical density of caspase-3 positive cardiac myofibrils, while increasing the heart tissue glutathione levels, compared to the ischemia-reperfusion alone group (p<0.01, p=0.011, and p=0.011, respectively).
Conclusion:
Whortleberry may be beneficial in preventing cardiac tissue damage caused by ischemia-reperfusion in the surgical repair of ruptured abdominal aortic aneurysms.
... We previously determined that the most effective concentration was 0.1 μm/L. 5,6 This current investigation focused on the effect of timing that PHC had on postconditioning. We also designed this in vitro experiment to further elucidate the mechanism of A/R injury in the early reoxygenation period. ...
... Our previous investigation showed that Penehyclidine Hydrochloride offered this protective effect via limiting calcium overload, decreasing formation of reactive oxygen species, restraining mitochondrial MPTP opening and reducing inflammatory response in vitro and in vivo. 5,6 The most effective dose of Penehyclidine Hydrochloride on H9c2 cell against anoxia/reoxygenation injury was determined to be 0.1 μm/L in our research group. 9 In 2012, Barrere-Lemaire et al found out the effective time window for delayed postconditioning treatment in rats model was 30 mins, 27 and our previous animal study determined the protective time window of PHC postcondition was within 10 mins after the reperfusion stage began. ...
... 9 In 2012, Barrere-Lemaire et al found out the effective time window for delayed postconditioning treatment in rats model was 30 mins, 27 and our previous animal study determined the protective time window of PHC postcondition was within 10 mins after the reperfusion stage began. 6 We hypothesized that IRI could be attenuated in the early period of the reperfusion stage. PHC was administered directly in the cell culture, which effected H9c2 cells directly, would offer more direct protective effect compared with those in vivo studies. ...
Background/Aims
To investigate the postconditioning protective effect of penehyclidine hydrochloride (PHC) against anoxia/reoxygenation (A/R) injury in H9c2 cells along with the involved mechanism and timing effect.
Methods
We divided H9c2 cells into 7 groups: control group, A/R group and PHC+A/R groups at 0 min, 5 mins, 10 mins, 20 mins, 30 mins, respectively (treated with 0.1 μm/L PHC at 0 min, 5 mins, 10 mins, 20 mins, 30 mins after the reoxygenation procedure began). Cell apoptosis, oxidative stress, intracellular Ca²⁺ concentration, mitochondrial membrane potential and mitochondrial permeability transition pore (MPTP) opening were explored. Bcl-2, Bax, Cyt C, caspase-3 and caspase-9 levels were measured.
Results
A/R significantly increased both cell injury and cell apoptosis. PHC showed postconditioning protective effect by attenuating superoxide production, decreasing Ca²⁺ overload, restraining MPTP activities, restoring mitochondrial membrane potential, regulating cell apoptosis proteins and modulation of mitochondrial pathway. Earlier administration of PHC offered greater postconditioning protective effect.
Conclusion
H9c2 cells were protected by PHC from A/R injury regardless of timing of PHC administration (0 min, 5 mins, 10 mins, 20 mins, 30 mins). However, earlier administration of PHC resulted in better PHC postconditioning protection.
... Apoptosis serves an important role in the progression of cerebral I/R injury (30). A previous study indicated that the expression level of caspase-3, Bax/Bcl-2 ratio and rate of cellular apoptosis were elevated during cerebral I/R (31). ...
... Ischemic hypoxia results in an increased number of free radicals during reperfusion, and such reperfusion stimulating extensive oxidative damage is considered one of the major reasons for brain cell damage and death in I/R injury (34). Tan et al (30) observed that oxidative stress was induced by cerebral I/R injury as indicated by suppressed SOD and GSH-Px levels, and increased MDA level (35). Numerous researchers have demonstrated the anti-oxidative effect of β-PAE. ...
β-patchoulene (β-PAE), an active constituent of the Pogostemon cablin, is well known for its anti-inflammatory and antioxidative functions in various diseases. However, little is known about the impact of β-PAE on the cerebral ischemia-reperfusion (I/R) injury. The current study aimed to determine the neuroprotective effect of β-PAE and the underlying mechanisms on cerebral I/R injury. Following pretreatment with β-PAE (10 mg/kg body weight) by tail intravenous injection for 1 h, Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 h and reperfusion for 24 h. The results indicated that pretreatment with β-PAE could diminish the infarct volume, decrease the brain water content, reduce the neurological deficit score and restore the mitochondrial membrane potential, compared with the untreated I/R injury group. Furthermore, cell apoptosis was markedly suppressed by β-PAE, and this effect was associated with the decreased apoptosis regulator BAX/apoptosis regulator Bcl-2 expression ratio and caspase-3 activity. In addition, β-PAE significantly inhibited the release of proinflammatory factors, including tumor necrosis factor-α, interleukin (IL)-1β and IL-6. Superoxide generation and malondialdehyde levels were reduced while the levels of glutathione peroxidase and superoxide dismutase were elevated following treatment with β-PAE, indicating the antioxidative role of β-PAE in cerebral I/R injury. Furthermore, the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway was inhibited by β-PAE, as demonstrated by the decreased TLR4 expression and nuclear translocation of p65, and increased IκBα level. Taken together, the results suggested that β-PAE may exhibit a neuroprotective effect on cerebral I/R injury in rats through inactivating the TLR4/NF-κB signaling pathway.
... Additional studies further indicate that PHC selectively blocks muscarinic acetylcholine (M) receptor M1, M3 and nicotinic acetylcholine receptors, with fewer M2 receptor-associated cardiovascular side effects than hyoscyamine (31). An increasing number of studies have indicated that PHC exhibits anti-apoptotic, anti-inflammatory and anti-oxidative stress effects under organ dysfunction (16,32,33). A previous study demonstrated that PHC exerted anti-inflammatory properties and protective effects during ALI via the inhibition of the toll-like receptor 4 signaling pathway (34). ...
Acute lung injury (ALI) is a critical syndrome that is associated with high morbidity and mortality rates. The activation of the Fas/Fas ligand (FasL) signaling pathway may be an important pathophysiological mechanism during ALI development. Penehyclidine hydrochloride (PHC) has been revealed to exhibit anti-apoptotic properties and may attenuate the observed systemic inflammatory response. The present study was performed to elucidate the molecular mechanism of PHC in the regulation of the Fas/FasL signaling pathway in rats with ALI. An ALI rat model was constructed by inducing blunt chest trauma and hemorrhagic shock (T/HS), with PHC administration prior to or following T/HS. At 6 h following T/HS, blood samples and lung tissues were collected. Western blotting, arterial blood gas analysis, ELISA, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and biochemical indicator analysis were performed to determine the degree of lung injury and the key signaling pathways associated with lung damage. The results indicated that the administration of PHC following T/HS effectively attenuates lung injury by improving pulmonary oxygenation, decreasing histopathological damage, decreasing polymorphonuclear neutrophil count and decreasing Fas, FasL, caspase-8, caspase-3, tumor necrosis factor-α, interleukin (IL)-6 and IL-1β expression. The results indicated that PHC exhibits anti-apoptotic functions and exerts protective effects in ALI rats induced by T/HS, which may be attributed to the inhibition of the Fas/FasL signaling pathway.
... Its antiapoptotic mechanism consists of suppression of Bax expression, activation of Bcl-2, promotion of the recovery of mitochondrial outer membrane permeabilization, and inhibition of voltagedependent anion channel 1 (VDAC1), cytosol cytochrome c (cyt-c), and cleaved caspase-3 in myocardial I/R. 30,31 This finding was confirmed by Lin et al, based on a study in H9c2 cells after anoxia/reoxygenation (A/R). Additionally, VDAC1 may be a bona fide target of PHC for protection against myocardial I/R injury. ...
Background
Penehyclidine hydrochloride (PHC) is an anticholinergic drug manufactured in China. It is used widely in clinics as a reversal agent in cases of organic phosphorus poisoning and as a preanesthetic medication. Compared with other anticholinergic agents, PHC confers substantial advantages. Here, in this review, we focus on its important clinical effects for organic phosphorus poisoning, preanesthetic medication, and the protective effects on certain visceral organs.
Materials and methods
Our bibliographic sources include the PubMed and China National Knowledge Infrastructure (CNKI) databases, updated in March 2018. To assess the data in detail, we used the search terms “penehyclidine hydrochloride,” “preanesthetic medication,” and “organic phosphorus.” Papers were restricted to those published in the English and Chinese languages, and to “paper” and “review” as the document type.
Results
PHC can effectively antagonize the symptoms of central and peripheral poisoning caused by organophosphorus poisoning. As a preanesthetic medication, it can not only effectively reduce mucus secretion and vascular infiltration but can also relax airway smooth muscles, dilate bronchioles in pulmonary conditions such as bronchiectasis, and increase pulmonary dynamic compliance. It can also prevent reflexive actions of the vagus nerve caused by excessive acetylcholine release such as abnormal airway contraction. Furthermore, it can strengthen sedation, bidirectionally regulate heart rate, and effectively inhibit respiratory secretions. In recent studies, PHC was shown to also have protective effects on various organs, such as the heart, lungs, brain, kidneys, intestines, and liver.
Conclusion
PHC has beneficial pharmacological properties used in the treatment of organophosphorus poisoning and as a preanesthetic medication for its few side effects. It also has protective effects on multiple organs, suggesting that PHC has extensive clinical application value which is worth further research. This review should be of help to those intending to research these topics further.
