The protective effects of Gentianella acuta against endoplasmic reticulum stress-induced myocardial apoptosis in a rat model of ischemic heart failure

doi:10.21203/rs.3.rs-1984524/v1

Download PDF

Research Article

The protective effects of Gentianella acuta against endoplasmic reticulum stress-induced myocardial apoptosis in a rat model of ischemic heart failure

https://doi.org/10.21203/rs.3.rs-1984524/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Gentianella acuta (Michx.) Hulten (G. acuta) has been widely used in Mongolian medicines for the treatment of cardiovascular diseases in Ewenki and Oroqen, Inner Mongolia autonomous region, China. The current exploration was developed to scrutinize the impact of G. acuta on proteins associated with endoplasmic reticulum (ER) stress and apoptosis in a rat model of ischemic heart failure. Echocardiography was employed for analyzing cardiac function, and both weight gain and serum indices were monitored in study subjects. In addition, heart tissue samples were subjected to hematoxylin-eosin, Masson’s trichrome, and Sirian red staining. ER stress- and apoptosis-related protein expression was additionally evaluated via Western blotting and immunohistochemical staining. Together, these analyses revealed that G. acuta was able to alleviate cardiac dysfunction, collagen deposition, and myocardial injury, ameliorating ER stress and protecting the myocardium through anti-apoptotic pathways.

G. acuta

Heart Failure

Endoplasmic Reticulum Stress

Apoptosis

G.acuta was able to alleviate cardiac dysfunction, collagen deposition, and myocardial injury in a model of ischemic heart failure.
G.acuta can protect rat myocardial cells against ER stress and protect the myocardium through anti-apoptotic pathways.
This article provide a foundation for further studies aimed at treating ischemic heart failure.

Heart failure (HF) is a terminal complication of many cardiac disorders, and it is currently a leading cause of global cardiovascular morbidity and mortality [1, 2]. Moreover, it is the most prevalent driver of hospitalization among elderly people, and accounts for 9.3% of cardiovascular disease-related mortality [3], with a forecast prevalence of 44% by 2035 in China [4].

Endothelial dysfunction can result from endoplasmic reticulum (ER) stress and associated apoptotic cell death induced in response to calcium overload, hypoxia, oxidative stress, or hyperglycemic conditions [5]. ER stress is also closely linked to inflammation, obesity, and cardiovascular disease [6, 7]. The ER is an organelle that is central to cellular homeostasis, facilitating the translation, transportation, and folding of many proteins. Under conditions of ER stress, the protective unfolded protein response (UPR) is engaged [8], enhancing protein folding and transportation while suppressing protein synthesis. However, when the cellular damage is too extensive, this response may fail to restore cellular homeostasis, instead triggering apoptotic death [9]. Excessive or persistent ER stress can occur in response to caspase 12-induced apoptosis [10].

G. acuta is commonly utilized in traditional Tibetan and Mongolian medicinal practices, and is distributed throughout northern China, Siberia, the Mongolian plateau, and far easter Russia [11]. The Oroqen and Ewenki people often chew or consume preparations made from G. acuta as a means of preventing or treating heart disease, with good reported curative efficacy [12,13]. The entirety of the G. acuta grass is utilized as a medicine which is widely used for the treatment of hepatitis, jaundice, fever, arrhythmia, coronary heart disease[12,14,15].

Numerous studies have shown that G. acuta and its active components have a protective effect on the heart. It was found that G. acuta significantly improved cardiac function and inhibited myocardial fibrosis through anti-oxidative, anti-apoptosis and anti-inflammation activities in an isoproterenol (ISO)-induced rat model of HF[16-19].However, the role and mechanism of G. acuta in IHF are not clear. Therefore, the aim of the present study was to investigate the protective effects of G. acuta in a rat model of IHF and whether the mechanism underlying G. acuta against IHF effects is telated to ER stress responses and myocardial apoptosis, which will provide evidence for clinical application.

2.1 G. acuta sample preparation

Dry whole G. acuta plant samples were purchased locally in Inner Mongolia and identified by the School of Pharmacy of Hebei College of Traditional Chinese Medicine. To prepare a G. acuta solution for further use, 200g of G. acuta was soaked in 3000ml distilled water for 20 minutes, refluxed 2 times, and the filtrates were then combined and concentrated to 760 mL, yielding 200g/780 mL of G. acuta decocted in water.

2.2 Reagents and instruments

An in situ terminal transferase labeling (TUNEL) apoptosis detection kit was obtained from Merck-Calbiochem, USA. Monoclonal rabbit anti-rat GRP-78, anti-caspase-12, anti-BNP, anti-eIF-2α, anti-phosphorylated eIF-2α (p-eIF-2α), and horseradish peroxidase-conjugated secondary antibodies were procured from Abcam (USA). An ECG-6511 electrocardiograph was purchased from Shanghai Optoelectronic Medical Electronic Instrument Co., Ltd., while a Vevo770 small animal Doppler echocardiograph was obtained from VisualSonics (Canada), and a CheniDoc XRS+ chemiluminescence imaging system was from Bio-rad (USA).

2.3 Experimental animals

In general, 60 specific pathogen-free male Sprague-Dawley (SD) rats, (9 weeks old, 300-350 g) were obtained from Hebei Yiweiwo Biotechnology Co. Ltd. (license number SCXK (Hebei) 2020-002).Experimental animal Quality Certificate No. : 20210433, License No. : SCXK9 (Ji) 2020-002. These animal studies were designed with consideration to principles of safety and fairness, and the experimental animals involved in the research process met the relevant national requirements for medical experimental animals. All animals were housed in a standard animal care facility (20℃ - 25℃, relative humidity: 40% - 70%, 12 h light/dark cycle) with 10 per ventilated cage and free access to food and water.

2.4 Heart failure model establishment and treatment:

Before the experiment, rats were adaptively fed for 1 week;.After weighing, rats were anesthetized via an intraperitoneal injection of 2% pentobarbital sodium (40 mg·kg-1) at 0.35mg·100g-1. Then, 9 rats were selected for inclusion in the sham operation group using a random number table, while 51 rats were used to establish an ischemic heart failure model. Animals were routinely disinfected, fixed in the supine position, subjected to oral endotracheal intubation under direct visualization, connected to an animal artificial ventilator with positive pressure artificial respiration (tidal volume: 3 ml/100 g, respiratory rate 60-70 breaths/min). The limb II lead electrocardiogram was then recorded. The chest of each animal was then disinctected and draped with towels, with a ~2 cm longitudinal incision being generated on the left edge of the sternum followed by the 0.5-1 cm blunt separation of pectoralis major. Through subsequent thoracotomy via ribs 4-5, the hear was thereby exposed. The left anterior descending coronary artery was ligated with 5-0 ophthalmic noninvasive suture, with animals in the sham group instead undergoing thread insertion without ligation. The heart was then reinserted, the chest was closed layer by layer, and suction was generated with a 10ml sterile syringe while closing the front of the chest to restore negative thoracic pressure. Endotracheal intubation was removed after spontaneous breathing had resumed, and prophylactic antibiotics were injected into the abdomen for 3 days postoperatively. St-segment elevation for > 30 min on ECG evaluation, myocardial paleness below the ligation, and decreased beat strength were all indicative of successful myocardial infarction modeling. Echocardiography was performed 30 min after coronary artery ligation, and heart failure was diagnosed accordingly, with animals being separated into experimental groups. Of these animals, 36 underwent successful modeling and were assigned to the model group and intervention (low, medium, and high dose G. acuta) groups using a random number table, with 9 per group. From day 2 post-modeling, G. acuta was administered intragastrically at different concentrations (0.3, 0.6, 1.2 g/kg) once per day for 21 days. Animals in the control and model group were instead given normal saline (10 mL/kg). For further details, see Figure 1.

2.5 Cardiac function tests

At 2 h following the final treatment, isofluorane was used to anesthetize rats, and they then underwent M-mode and two-dimensional electrocardiography and echocardiography with Doppler echocardiography. Three consecutive values were recorded for the left ventricular ejection fraction (LVEF), and heart rate (HR) for each rat, with average values for each cardiac cycle being recorded along with a representative electrocardiogram from each group.

For ECG analyses, rats were anesthetized using 3% pentobarbital sodium (i.p.) and fixed to the operating surface in a supine position. Electrode needles were then inserted subcutaneously into each of the limbs of the subject, with ECG recordings then being made following lead wire connection.

2.6 Tissue staining and analysis

After analyzing cardiac functional indicators, rats were euthanized and the cardiac tissue was quickly removed, with the left ventricular anterior wall being fixed in 4$ neutral formaldehyde, dehydrated, paraffin-embedded, and cut into 4 μm thick segments which were used for immunohistochemical (IHC), hematoxylin and eosin (H&E), Masson’s trichrome, Sirian red, and TUNEL staining to assess changes in myocardial tissue pathology, as detailed below.

2.6.1 IHC staining: GRP-78, caspase-12, BNP, eIF-2α, and p-eIF-2α levels were detected in myocardial tissues via IHC staining. Prepared paraffinized tissue sections were heated for 2 h at 60℃, deparaffinized using xylene I and II (10 min each), and then rehydrated with an ethanol gradient (anhydrous ethanol, anhydrous ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol for 5 min each), washed with distilled water, subjected to heat-based antigen retrieval, processed with 3% H₂O₂ to block endogenous peroxidase activities, blocked with goat serum, incubated overnight with primary antibodies (1:1000) at 4℃, and then probed for 1 h with a universal secondary antibody. DAP was used for color development until yellow-brown particles were detected under microscopic examination. Nuclei were counterstained with hematoxylin, after which samples were dehydrated, treated to transparency, and mounted using neutral gum prior to microscopic imaging and analysis.

2.6.2 H&E staining: Tissue sections were deparaffinized, after which nuclei were stained by employing Harris’ hematoxylin for 3-8 min. Cells were then rinsed using tap water, differentiated for a few seconds in HCl, treated with 0.6% aqueous ammonia to promote blue color development, and rinsed under running water. The cytoplasm was stained by treating samples with eosin staining solution for 1-3 min, after which samples were dehydrated (95% ethanol, 95% ethanol, anhydrous ethanol, anhydrous ethanol, xylene, xylene, 5 min each), mounted with neutral gum, and microscopically imaged for subsequent analysis.

2.6.3 Masson’s staining: Tissues were deparaffinized and rinsed by employing faucet water and distilled water, after which nuclei were stained for 5-10 min with Regaud hematoxylin stain or Weigert hematoxylin. Samples were then washed, differentiated with HCl and ethanol if overstained, rinsed with water, and treated for 5-10 min followed by soaking in a 2% glacial acetic acid aqueous solution and treating for 3-5 min with 1% phosphomolybdic acid aqueous solution for differentiation. Samples were then directly dyed for 5 min with aniline light green or blue solution, soaked again in 0.2% glacial acetic acid, and treated with 95% ethanol, anhydrous ethanol, xylene, and sealed with neutral gum before microscopic imaging and analysis.

2.6.4 Sirian red staining: Samples were deparaffinized, stained for 10-20 min with Weigert iron hematoxylin staining solution, and differentiated for a few seconds using acidic differentiation solution. Samples were then rinsed for 5-10 min with faucet water succeeded by further rinsing with distilled water. Samples were then stained with Sirius red staining solution for 1 h, rinsed under running water, dehydrated and treated to transparency, sealed with neutral gum, and imaged via microscope for subsequent analysis.

2.6.5 TUNEL staining: Segments were deparaffinized, rehydrated, and treated with proteinase K and 3% H₂O₂. Then, 50 μl of TUNEL reaction solution was dropwisely added, and samples were incubated for 1 h at 37°C, washed, and 50 μl of Streptavidin-HRP working solution was dropwisely added followed by a 20 minute incubate in the dark. DAB dilution solution was then dropwisely added to facilitate color development, which was terminated by adding ddH₂O. Nuclei were stained for 2 min with hematoxylin, followed by treatment for 20 s with lithium carbonate. Samples were then mounted and the apoptotic cells (with brown nuclei) in 5 random fields of view per sample were counted under the microscope. The cardiomyocyte apoptosis rate was then calculated based on the ratio of apoptotic cells to total cells.

2.7 Western blotting

Samples of left ventricular anterior wall tissue from individual rats were collected, ground in liquid nitrogen, and lysed on ice for 25 min succeeded by centrifugation for 15 min at 12,000 rpm (centrifuge radius: 12 cm). Supernatant protein levels were then detected via BCA assay, with 40 μg of protein per sample then being separated via SDS-PAGE and transferred onto an appropriate membrane. Blots were blocked for 2 h at ambient temperature, succeeded by an incubation during the night hours with primary antibodies specific for GRP-78 (1:500), caspase-12 (1:500), BNP (1:1000), eIF-2α (1:1000), or p-eIF-2α (1:1000) at 4℃ with shaking. Blots were washed thrice with TBST (TBS + 0.5% Tween), then incubated for 2 h with secondary antibody (1:10,000) at room temperature, washed again, and developed in a darkroom. Images were collected emloying a gel imaging system, and densitometric analyses were performed with the image analysis computer program, with GAPDH serving as a normalization control.

2.8 Statistical analysis:

All findings were scrutinized employing SPSS 20.0. Normally distributed outcomes are given as the mean ± standard deviation, and were studied through single-factor ANOVAs, with SNK q tests being used for pairwise sample comparisons. P < 0.05 was the significance threshold.

3.1 The impact of G. acuta on body weight and serum parameters in a rat model of ischemic heart failure.

We began by evaluating changes in body weight over a 21-day period in our rat model of ischemic heart failure (Fig. 2). Regarding the group of control, the lowest levels of weight gain were observed in the model group, with increased weight gain in the high-, medium-, and low-dose G. acuta groups of treatment although these levels were less than those in control rats (P < 0.05).

Relative to the control group, serum LDH, and CK levels were all substantially elevated in the model group, whereas they were significantly reduced in all three G. acuta treatment groups as compared to the model group (all P < 0.05; Fig. 3). There existed no considerable discrepancies in these levels between the low and medium dose level groups (P > 0.05).

3.2 G. acuta protects against ischemic heart failure-related cardiac dysfunction.

Next, echocardiography and ECG analyses were used to explore cardiac function in our experimental rats as a means of gauging the protective activity of G. acuta as a bulwark against myocardial damage. In ECG analyses, model group rats exhibited a tall T wave, ST-segment elevation, and an R wave decrease relative to the control group, whereas all three G. acuta treatment groups exhibited a reversal of these changes, with the phenotype of rats in the high-dose group being similar to that in sham control rats (Fig. 4).

Echocardiography (Fig. 5a)revealed significant decreases in the EF of rats in the model group in comparison to the control group, whereas HR exhibited the opposite trend (P < 0.05)(Fig. 5b). Relative to the model group, HR was remarkably decreased and EF were significantly increased in the three G. acuta treatment groups (P < 0.05) in a dose-dependent behavior, with no substantial discrepancies between the low- and medium-dose groups (P > 0.05).

3.3 G. acuta protects against myocardial damage in a rat model of ischemic heart failure.

To assess the protective efficacy of G. acuta treatment against pathological damage to the myocardium in our experimental rats and to evaluate concomitant myocardial fibrosis, we next conducted H&E, Masson’s trichrome, and Sirian red staining of the cardiac tissue from the experimental animals.

Relative to control group rats, those in the model group exhibited extensive myocardial cell necrosis and neutrophilic infiltration, with observed myocardial lesions primarily being localized within the endocardial and middle layers of the myocardium. The observed myocardial injuries were additionally determined through multiple focal necroses and myofibril deposition with concomitant inflammatory cell infiltration. These lesions were less pronounced in the samples from the G. acuta treatment group, with reductions in necrotic area, inflammatory cell infiltration, and myofibril deposition as compared to model group rats (Fig. 6a). Masson’s trichrome staining and Sirian red staining further confirmed an increase in left ventricular collagen deposition in the model group, whereas this effect was reversed by G. acuta treatment. Together, these data suggest that G. acuta can attenuate damaging cardiac structural alterations and fibrosis in a rat model of ischemic heart failure (Fig. 6b-c).

3.4 G. acuta inhibits myocardiocyte apoptosis in a rat model of ischemic heart failure.

TUNEL staining of myocardial tissue samples from these rats was next performed (Fig. 7a), revealing myocardial cell apoptosis rates of 12.5 ± 00.66%, 88.9 ± 0.85%, 76.4 ± 1.41%, 54.1 ± 1.40%, and 31.1 ± 1.15%, respectively, in the sham control, model, low-dose, medium-dose, and high-dose treatment groups, with significant differences among these groups. Specifically, the cardiomyocyte apoptosis rate was significantly lower in the three G. acuta treatment groups relative to the model group (P < 0.01), however these rates remained higher than those in the sham group, with rates decreasing in a dose-dependent behavior in response to G. acuta treatment (P < 0.01) (Fig. 7b).

3.5 Analysis of apoptosis-related protein expression in rat cardiomyocytes.

To gain further insight into the mechanisms whereby G. acuta alters apoptotic cell death in rat cardiomyocytes, we next conducted IHC staining and Western blotting analyses. IHC staining revealed that there were significant reductions in caspase-12 and BNP protein accumulation in the three G. acuta treatment groups as compared to the model group (Fig. 8a and b). Western blotting further confirmed these results, revealing that the observed increases in caspase-12 and BNP in the model group were considerably decreased in response to G. acuta treatment (P < 0.05 or P < 0.01)(Fig. 9a and b). Overall, these findings confirmed the ability of G. acuta to suppress apoptosis-associated protein expression within rat cardiomyocytes.

3.6 Analysis of myocardial proteins in the myocardium of rats associated with ER stress.

To further explore the effects of G. acuta on ischemic heart failure, IHC staining and Western blotting were further employed for analyzing the expression of GRP-78 and p-eIF-2α. Significant positivity for both of these proteins was observed in the model group, whereas lower levels of GRP-78 and p-eIF-2α accumulation were observed in the three treatment groups (Fig. 8c and d). Western blotting was additionally used for semiquantitative analyses of GRP-78 and p-eIF-2α levels. In line with the IHC results, GRP-78 and p-eIF-2α protein expression were remarkably enhanced in the model group (P < 0.01) and significantly reduced in response to all three tested G. acuta doses (P < 0.05 or P < 0.01)(Fig. 9c and d). These data thus confirmed the ability of G. acuta to reduce the myocardial levels of proteins associated with ER stress.

Cardiovascular diseases are among the fundamental global reasons for morbidity and mortality [20]. Heart failure is the terminal stage of several cardiovascular diseases including hypertension, cardiomyopathy, valvular heart disease, and ischemic heart disease, and it is associated with a case fatality rate of ~ 4.1% [21]. The mechanistic basis for HF is highly complex, making the identification of novel approaches to treating this condition particularly pressing. Efforts to understand the pathogenesis of this condition and to protect myocardial cells are thus an area of active research. Herein, we established a rat model of ischemic heart failure and used these animals to explore the protective effects of G. acuta on cardiac function in addition to exploring the underlying mechanisms whereby this herb mediates its beneficial effects.

ER stress and the UPR have previously been identified as important mechanisms induced in response to ischemia/reperfusion injuries [22, 23]. The ER is a multifunctional organelle that regulates protein folding, synthesis, and transport, and the dysregulation of these processes can thus contribute to ER stress [24]. The UPR can be induced to restore ER functionality, but when this response fails to restore ER homeostasis, excess protein aggregation can contribute to the induction of cell death [10]. GRP78 upregulation is indicative of ER stress. Under basal conditions, GRP78 remains inactive in a complex with PERK [25]. When unfolded proteins accumulate within the ER, however, GRP78 dissociated from PERK which is then phosphorylated and activated, thus inducing the UPR, promoting eIF2α phosphorylation, and driving selective ATF4 translation [26]. ATF4 can then coordinate key aspects of the UPR, promoting enhanced amino acid metabolism, redox reactions, other stress responses, and the modulation of ER stress-related target genes that influence protein secretion. When stressful conditions are maintained for extended periods, however, ATF4 can instead promote the expression of CHOP, which is a pro-apoptotic gene [27]. The upregulation of CHOP performs a fundamental task in the induction of apoptosis, which is controlled by multiple regulatory pro- and anti-apoptotic proteins, including caspase-12 [28–32].

Caspase-12 is a caspase-aspartase family protein that is ER-specific and involved in the non-mitochondria-dependent induction of apoptotic death in the context of ER stress, making it a key regulator of this process in cardiomyocytes. At baseline, caspase-12 creates a complex with tumor necrosis factor receptor-associated factor 2 (TRAF2) and remains in an inactive state. In the context of severe ER stress, however, caspase-12 can be dissociated and activated, contributing to apoptotic induction [33].

In the present analysis, ER stress-related proteins (GRP-78 and p-e1F2α) were analyzed via IHC and Western blotting, revealing both to be significantly upregulated in our rat model of ischemic heart failure. Prior reports have suggested that GRP-78 upregulation and e1F2α phosphorylation help to protect cells against ER stress, suggesting the induction of protective responses in our model rats. G. acuta treatment, in contrast, was associated with reduced expression of these proteins consistent with the ability of such treatment to protect against ER stress induction and associated cardiac dysfunction. G. acuta may thus selectively act against GRP-78 and promote e1F2α dephosphorylation, making it a promising new therapeutic agent for use in treating heart failure. We additionally analyzed caspase-12 as a readout for apoptotic activity in the myocardial tissue of our model rats, and observed decreases in caspase-12 levels and associated apoptotic death in G. acuta-treated rat myocardial tissue. Overall, these data thus indicate that G. acuta can mitigate both ER stress and caspase-12 expression to protect cardiomyocytes against apoptotic death.

Cardiac remodeling occurs as a consequence of imbalanced extracellular matrix (ECM) synthesis and degradation. As cardiomyocytes are terminally differentiated and incapable of proliferation, their apoptotic death results in an overall reduction in cardiomyocyte numbers and the compensatory production of excessive ECM components such as collagen. The resultant imbalance contributes to myocardial fibrosis, and remodeling, ultimately resulting in decompensated heart failure [34, 35]. As such, myocardial apoptosis is a key driver of myocardial remodeling. Moreover, cardiomyocytes are highly metabolically active cells and are thus extremely sensitive to ER stress and associated inflammation [36], placing them at a high risk of such apoptotic death.

Briefly, the achievements of this research indicate that G. acuta can protect rat myocardial cells against ER stress and apoptotic death, thereby preventing cardiac dysfunction in a model of ischemic heart failure (Fig. 10). These findings provide a foundation for further studies aimed at treating heart failure.

All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

Ethics approval and consent to participate.

All experiments were conducted in accordance with the P.R. China legislation for the use and care of laboratory animals. All animal experiments were approved by the ethics committee of Hebei University of Chinese Medicine (certifcate number DWLL2018026, 18 Dec, 2018).

Consent for publication

Not applicable.

Availability of data and materials

This manuscript is guaranteed to be original.The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Funding

This work was supported by the National Natural Science Foundation of China（Grant no. 81500317 ), the Natural Science Foundation of Hebei Province（Grant no. H2017423033 , H2020423061), the science and technology research projects in universities of Hebei Province（Grant no. ZD2017056 ).Authors' contributions

Acknowledgements

Not applicable.

Competing interests

The authors declare no competing interests.The article is not subject to the conflict of objective economy, personal interests and beliefs, and there is no potential competitive interests.

Heart Failure Group of Cardiovascular Medicine Branch, Chinese Medical Doctor Association , Expert Committee of Chinese Heart Failure Center Alliance. Expert consensus on clinical performance and quality measures for adults with heart failure in China. Chin J Med Sci Front (Electron V er). 2021;13:52-62.
Yancy CW, Jessup M, Bozkurt B, Butler J, Casey Jr DE, Colvin MM, et al. 2017 ACC/AHA/HFSA focused update of the 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. Journal of the American College of Cardiology. 2017;70:776-803.
Tomasoni D, Adamo M, Anker MS, von Haehling S, Coats AJS , Metra M. Heart failure in the last year: progress and perspective. ESC Heart Fail. 2020;7:3505-3530.
Hao Guang, Wang Xin, Chen Zuo, et al. Prevalence of heart failure and left ventricular dysfunction in China: the China Hypertension Survey, 2012-2015.European Journal of Heart Failure.2019;21(11):1329-1337.
Kim Inki, Xu Wenjie, Reed John C.Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities.Nature Reviews Drug Discovery.2008;7(12):1013-30.
Yao Fan, Zhang Ming, Chen Li. 5'-Monophosphate-activated protein kinase (AMPK) improves autophagic activity in diabetes and diabetic complications. Acta Pharmaceutica Sinica B.2016;6(1):20-5.
Ozcan Umut, Yilmaz Erkan, Ozcan Lale, et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science. 2006;313(5790):1137-40.
Wang Miao, Kaufman Randal J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature. 2016;529(7586):326-35.
Hetz Claudio, Chevet Eric, Harding Heather P. Targeting the unfolded protein response in disease. Nature Reviews Drug Discovery. 2013;12(9):703-19.
Szegezdi Eva, Logue Susan E, Gorman Adrienne M, et al. Mediators of endoplasmic reticulum stress-induced apoptosis. Embo Reports. 2006;7(9):880-5.
Wang Zhibin, Wu Qiong, Yu Ying, et al. Determination and pharmacokinetic study of four xanthones in rat plasma after oral administration of Gentianella acuta extract by UHPLC-ESI-MS/MS. Journal of Ethnopharmacology. 2015;174:261-9.
Wunir C. Khasbagan: Ewenki folk medicinal plants and its comparison with Mongolian medicine. Chin J Ethnomed Ethnopharm. 2009;18:156-158.
Wang Zhibin, Wu Gaosong, Liu Hua, et al. Cardioprotective effect of the xanthones from Gentianella acuta against myocardial ischemia/reperfusion injury in isolated rat heart. Biomedicine & Pharmacotherapy. 2017;93:626-635.
Ding Zhijuan, Liu Yanxia, Ruan Jingya, et al. Bioactive Constituents from the Whole Plants of Gentianella acuta (Michx.) Hulten. Molecules (Basel, Switzerland).2017;22(8).
Liu Yanxia, Ni Yajuan, Ruan Jingya, et al. Bioactive gentixanthone and gentichromone from the whole plants of Gentianella acuta (Michx.) Hulten. Fitoterapia. 2016;113:164-9.
Li Ai-Ying, Wang Jing-Jing, Yang Sheng-Chang, et al. Protective role of Gentianella acuta on isoprenaline induced myocardial fibrosis in rats via inhibition of NF-κB pathway. Biomedicine & Pharmacotherapy. 2019;110:733-741.
Sun Jia-Huan, Yang Hong-Xia, Yao Ting-Ting, et al. Gentianella acuta prevents acute myocardial infarction induced by isoproterenol in rats via inhibition of galectin-3/TLR4/MyD88/NF-кB inflammatory signalling. Inflammopharmacology. 2021;29(1):205-219.
Yang Hong-Xia, Xu Geng-Rui, Zhang Chuang, et al. The aqueous extract of Gentianella acuta improves isoproterenol-induced myocardial fibrosis via inhibition of the TGF‑β1/Smads signaling pathway. International Journal of Molecular Medicine. 2020;45(1):223-233.
Xu Geng-Rui, Zhang Chuang, Yang Hong-Xia, et al. Modified citrus pectin ameliorates myocardial fibrosis and inflammation via suppressing galectin-3 and TLR4/MyD88/NF-κB signaling pathway. Biomedicine & Pharmacotherapy. 2020;126:110071.
Shen Chengxing, Ge Junbo. Epidemic of Cardiovascular Disease in China: Current Perspective and Prospects for the Future. Circulation. 2018;138(4):342-344.
Zhang Yuhui, Zhang Jian, Butler Javed, et al. Contemporary Epidemiology, Management, and Outcomes of Patients Hospitalized for Heart Failure in China: Results From the China Heart Failure (China-HF) Registry. Journal of Cardiac Failure. 2017;23(12):868-875.
Qiu Jian, Wang Xin, Wu Fei, et al. Low Dose of Apelin-36 Attenuates ER Stress-Associated Apoptosis in Rats with Ischemic Stroke. Frontiers in Neurology. 2017;8:556.
Lin Yu Wen, Chen Tsung Ying, Hung Chia Yang, et al. Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress. International Journal Of Molecular Medicine. 2018;42(1):182-192.
Iurlaro Raffaella, Muñoz-Pinedo Cristina. Cell death induced by endoplasmic reticulum stress. FEBS Journal. 2016;283 (14):2640-52.
Ibrahim Ibrahim M, Abdelmalek Doaa H, Elfiky Abdo A. GRP78: A cell's response to stress. Life Sciences. 2019;226:156-163.
Rozpedek W, Pytel D, Mucha B, et al. The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. Current Molecular Medicine. 2016;16 (6):533-44.
Yang Wei, Paschen Wulf. Unfolded protein response in brain ischemia: A timely update.Journal Of Cerebral Blood Flow and Metabolism. 2016;36(12):2044-2050.
Chen Yifang, Gui Dingkun, Chen Jianguo, et al. Down-regulation of PERK-ATF4-CHOP pathway by Astragaloside IV is associated with the inhibition of endoplasmic reticulum stress-induced podocyte apoptosis in diabetic rats. Cellular Physiology and Biochemistry. 2014;33(6):1975-87.
Hu Yimeng, Liu Jie, Yuan Yin, et al. Sodium butyrate mitigates type 2 diabetes by inhibiting PERK-CHOP pathway of endoplasmic reticulum stress. Environmental Toxicology and Pharmacology. 2018;64:112-121.
Ljubkovic Marko, Gressette Melanie, Bulat Cristijan, et al. Disturbed Fatty Acid Oxidation, Endoplasmic Reticulum Stress, and Apoptosis in Left Ventricle of Patients With Type 2 Diabetes. Diabetes. 2019;68 (10):1924-1933.
Chen Ming-Tai, Huang Ruo-Lan, Ou Li-Jun, et al. Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway. Chinese Journal of Integrative Medicine. 2019;25(8):604-612.
Wang Yan, Xue Jingjing, Li Yan, et al. Telmisartan protects against high glucose/high lipid-induced apoptosis and insulin secretion by reducing the oxidative and ER stress. Cell Biochemistry and Function. 2019;37(3):161-168.
Liu Hua, Wang Zhe, Nowicki Michael J. Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice. World Journal of Gastroenterology. 2014;20(48):18189-98.
Rani Shilpa, Sreenivasaiah Pradeep Kumar, Cho Chunghee, et al. Salubrinal Alleviates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Endoplasmic Reticulum Stress Pathway. Molecules and Cells. 2017;40(1):66-72.
Pei Zhaohui, Deng Qinqin,Babcock Sara A, et al. Inhibition of advanced glycation endproduct (AGE) rescues against streptozotocin-induced diabetic cardiomyopathy: Role of autophagy and ER stress. Toxicology Letters. 2018;284:10-20.
Zhang Cheng, Syed Taha Wasim, Liu Renjing, et al. Role of Endoplasmic Reticulum Stress, Autophagy, and Inflammation in Cardiovascular Disease. Frontiers in cardiovascular medicine. 2017;4:29.

No competing interests reported.

WesternBlotoriginaldata.ppt

Download PDF

Editorial decision: Major revision
06 Jul, 2023
Reviews received at journal
27 Jun, 2023
Reviewers agreed at journal
06 Jun, 2023
Reviewers agreed at journal
26 Mar, 2023
Reviews received at journal
31 Jan, 2023
Reviewers agreed at journal
29 Jan, 2023
Reviewers invited by journal
23 Sep, 2022
Editor assigned by journal
23 Sep, 2022
Editor invited by journal
22 Sep, 2022
Submission checks completed at journal
22 Sep, 2022
First submitted to journal
21 Aug, 2022

You are reading this latest preprint version

The protective effects of Gentianella acuta against endoplasmic reticulum stress-induced myocardial apoptosis in a rat model of ischemic heart failure

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1 G. acuta sample preparation

2.2 Reagents and instruments

2.3 Experimental animals

2.4 Heart failure model establishment and treatment:

2.5 Cardiac function tests

2.6 Tissue staining and analysis

2.7 Western blotting

2.8 Statistical analysis:

3. Results

3.1 The impact of G. acuta on body weight and serum parameters in a rat model of ischemic heart failure.

3.2 G. acuta protects against ischemic heart failure-related cardiac dysfunction.

3.3 G. acuta protects against myocardial damage in a rat model of ischemic heart failure.

3.4 G. acuta inhibits myocardiocyte apoptosis in a rat model of ischemic heart failure.

3.5 Analysis of apoptosis-related protein expression in rat cardiomyocytes.

3.6 Analysis of myocardial proteins in the myocardium of rats associated with ER stress.

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1