Total saponins from Trillium Tschonoskii Maxim alleviate myocardial ischemia reperfusion injury in rat

Background (TST) on myocardial ischemia reperfusion injury in rat.

The mechanism of MIRI is very complex, which mainly include oxidative stress, calcium overload, energy metabolism disorder, in ammatory response, mitochondrial cell apoptosis and so on [11][12][13]. Relevant statistics show that nearly 1/4 of patients cannot provide enough normal myocardium after myocardial ischemia-reperfusion treatment, resulting in serious heart failure [14][15][16]. Therefore, the mechanism study of blood reperfusion injury has become the focus in the research of myocardial ischemia. Some studies have shown that the protective agent of cardiomyocytes administrated before myocardial ischemia-reperfusion has a signi cant inhibitory effect on myocardial injury [17][18][19], which is of great signi cance for the prevention of MIRI. Therefore, it is very important to nd effective drugs for the treatment of MIRI.
Trillium tschonoskii Maxim. (TTM), a plant from Trillocerosaceae family, is locally known as 'Yan Ling Cao', which meaning that it can prolong human life [20,21]. As a traditional Chinese medicinal plant in mid-western part of China, the dried root and rhizome of TTM were traditionally used for the treatment of hypertension, dizziness, neurasthenia, waist leg pain, bleeding, headache, and traumatic haemorrhage [22]. There have reported many literatures of TTM about in vivo animal studies, such as TTM extracts could mitigates D-galactose-induced brain aging of rats [21], attenuates abnormal Tau phosphorylation in rats [20], anti-tumor in non-small cell lung cancer in mice [23,24], reduce myocardial injury in diabetic rats [25], decrease liver injury in rats [26], and etc. Phytochemical studies revealed that TTM contains many of saponins [27][28][29], saponin, as the major bioactive component of TTM, were found to possess various bioactivities, for example anti-in ammatory, immune-improving, anti-aging, anti-oxidative, anti-bacterial, anti-virus and anti-tumor properties [30,31]. Although the saponins isolated from TTM showed various bioactivities, the reported pharmacological activities on MIRI were limited.
Furthermore, it was reported that the activation of adenosine 5'-mono phosphate activated protein kinase (AMPK) / silent information regulator 1 (Sirt1) signaling pathway could inhibited MIRI through alleviate in ammation reaction [32][33][34][35]. Thus, in this study, we attempted to explore and evaluate the protective effect and potential mechanisms of TST in MIRI in rat. In addition, based on the literature reported about AMPK / Sirt1 signaling pathway on MIRI, here, we also would like to know if TST have the protective effects on MIRI, is it related with AMPK / Sirt1 signaling pathway? The study would provide the experimental evidence for further clinical therapy of MIRI by TST.

Preparation of TST [36]
The root and rhizome of TTM was purchased from Enshi Chinese Medicine Co. Ltd. ( Take about 10 kg of air-dried plant materials, crushed into powder and through 60 mesh sizes. Soak the powder with 70% ethanol, heat and re ux for 3 hours with three times (1:8 for the rst extraction and 1:6 for the second and third extraction, w/v for drug powder/ethanol solvent), then ltered. Next, the drug ltrates were evaporated and concentrated to remove ethanol under vacuum. Suspend the concentrated drug extract with distilled water and then add petroleum ether and n-butanol successively to partition, the n-butanol extract was considered as TST. Finally, collected the n-butanol extract, concentrated, vacuum dried, and nally got TST. The content of TST was 19.1 mg/g (based on crude drugs).

Animals
Forty-eight male Sprague Dawley (SD) rats with body weight 200±20 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK 2009-0011, Beijing, China). Six rats were kept in one polyacrylic cages. The rats were free access to food and water, housed in the standard controlled conditions with the temperature in 23 ± 2°C, the humidity in 50 ± 5%, and a 12 h day/night cycle. The rats were quarantined for one week before the experiments. The human care of the rats was according to the National Institutes of Health Guidelines of China and related ethical regulations of Beijing University of Chinese Medicine (Beijing, China).

Experimental design
All the rats were raised for one week before the formal experiment, in order to adapt the environment. The rats were randomly divided into four groups (n=12): Sham group, ischemia reperfusion injury (IR) group, TST-L 100 mg/kg group and TST-H 200 mg/kg group. The rats in TST group were oral administrated with the related dosage of TST for 14 days. The rats in Sham and IR group were oral administrated with normal saline for 14 days. After drug administration, the rats except Sham group were established IR model according to the method of Xu et al. reported [37]. The ischemic time is 30 min, the reperfusion time is 120 min. The rats in Sham group were surgically as IR rats except the suture was in the coronary artery without ligation. At the end of the experiment, all the rats were sacri ced by cervical dislocation.

Hemodynamic indexes detection
After myocardial ischemia and reperfusion, all the rats were detected the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVDP), the maximum rate of left ventricular pressure rise (+dp/dt max ) and the maximum rate of decline (-dp/dt max ) by BL420F physiological instrument.

Anti-oxidative index detection
Heart homogenate (10%, w/v) was prepared by homogenizing the heart tissue in 150 mM Tris-HCl buffered saline (pH 7.2) with a polytron homogenizer. The enzyme activity of SOD and GSH-Px in heart tissue was measured according to the commercial kit. The data are read at the wavelength of 550 nm and expressed as U/L and nmol/L respectively. The lipid peroxidation products MDA in heart tissues was measured at 532 nm according to the protocol of commercial kit. The data of MDA was expressed as μmol/g protein.

In ammatory factors detection
The levels of TNF-α, IL-1β and IL-6 in heart tissue homogenate were measured by ELISA kit from BioVision following the protocol provided by manufacture. The data were obtained at the wavelength of 450 nm and expressed as pg/mL. The preparation of heart tissue homogenate was followed the section of "Anti-oxidative index detection".

Histopathological examination
Heart specimens were xed in 4% neutral formaldehyde buffer for overnight, and then embedded in para n, cut into 5 µm thickness for hematoxylin and eosin (H&E) staining. The H&E sections were examined and photographed under a Olympus BX-50 light microscope at 200× magni cation.

TUNEL staining
For terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, the hearts of rats were xed in 4% paraform-aldehyde and dehydrated in 20% sucrose, embedded in para n and sectioned (5 μm). The sections were treated as indicated in the situ cell death detection kit (Nanjingjiancheng, China). Thereafter, nuclei were co-stained with hematoxylin.

Statistical analysis
The biological data presented as mean ± SD. The statistical comparisons were made by One-way ANOVA test followed by Dunett's t-test with GraphPad Prism 6.0 statistical software. P < 0.05 and P < 0.01 showed a statistically signi cant.

Hemodynamic changes of rats in each group
Compared with Sham group, the levels of LVSP and + dp/dt max were signi cantly decreased, while the levels of LVDP and -dp/dt max were signi cantly increased in the rats of IR group (P < 0.01, Table 1), this phenomenon exhibited that MIRI can damage the contractile and diastolic ability of myocardium. Compared with IR group, the levels of LVSP and + dp/dt max were markedly increased, and the levels of LVDP and -dp/dt max were signi cantly decreased in the rats of TST-treated groups (P < 0.05, P < 0.01, Table 1), which suggested that TST can effectively improve the hemodynamic indexes in IR rats.

Effects of TST on anti-oxidative indexes
From Table 2, we can see that the levels of SOD and GSH-Px were signi cantly decreased in IR group, compared with Sham group (P < 0.01). Pre-treatment with 100 and 200 mg/kg TST, the levels of SOD and GSH-Px were signi cantly increased (P < 0.05, P < 0.01), compared with IR group. The contents of MDA in heart tissue was signi cantly increased in IR group, compared with Sham group (P < 0.01). While the rats in TST pre-treated group were markedly decreased the contents of MDA, compared with that of IR group (P < 0.05, P < 0.01).

Effect Of Tst On Macrophage In ltration
In Fig. 1, macrophages in heart tissue are dark brown. In the sham operation group, there were a few macrophages in the heart tissue, while in IR model group, there were a lot of macrophage in ltration. Compared with IR model group, the number of macrophages were signi cantly decreased in the heart tissue of low-dose TST-L rats, while only a small amount of macrophages were in ltrated in the heart tissue of high-dose TST-H rats.

Effects of TST on in ammatory factors
The in ammation of the heart tissue were evaluated by the levels of TNF-α, IL-1β and IL-6. Compared with Sham group, the levels of TNF-α, IL-1β and IL-6 were remarkably increased in IR group (P < 0.01, Table 3). TST pre-treated group were signi cantly decreased the levels of TNF-α, IL-1β and IL-6, compared with IR group (P < 0.05, P < 0.01, Table 3).

Discussion
Myocardial ischemic disease is one of the main diseases endangering human health [38]. The clinical therapy method of thrombolysis, percutaneous coronary angioplasty, and coronary artery bypass grafting can restore blood supply and improve heart function in a short time, but still cannot avoid the aggravation of tissue damage during ischemia-reperfusion, or even lead to irreversible function and organic damage [39]. The mechanism of MIRI is currently considered to be related to the role of free radicals, intracellular calcium overload, leukocyte activation, endoplasmic reticulum stress and mitochondrial dysfunction. Among them, reactive oxygen species (ROS) damage is considered to be important for MIRI [40]. Many studies have shown that excessive release of oxygen free radicals can induce irreversible pathological changes of myocardial cells, thus aggravating the reperfusion injury of blood ow [41]. In this study, through the pathological observation of myocardial tissue in the model group, we found that the necrosis and edema of myocardial tissue make the in ammatory in ltration of myocardial cells.
In this study, the levels of LVSP and + dp/dt max in IR group were signi cantly increased, LVDP anddp/dt max were markedly decreased, compared with Sham group, which indicating that MIR can lead to myocardial necrosis. While the myocardial hemodynamics of the rats pre-treated with TST were signi cantly improved, indicating that TST can improve and protect the myocardial structure and function, improve the pathological changes and inhibit the MIRI.
In oxidative stress, MDA can indicate the degree of lipid peroxidation and the degree of damage of oxygen free radicals to cardiomyocytes, while SOD and GSH-Px can clear these oxygen free radicals and degrade them. Therefore, the activity of SOD and GSH-Px shows the ability of myocardial cells to scavenge oxygen free radicals [42,43]. In the study, the activity of SOD and GSH-Px were signi cantly decreased, while the contents of MDA was markedly increased in IR group. TST pre-treatment can signi cantly increase the enzyme activity of SOD and GSH-Px, and signi cantly decrease the MDA products. This results exhibited that TST can increase the anti-oxidative ability of cardiomyocytes, further to protect the MIRI rats, decrease the injury.
In addition, in the study, we detected the protein expression of caspase-12, caspase-9, and caspase-3 in classical signaling pathway of apoptosis by western blot method. The result showed that TST treatment group signi cantly reduced the apoptosis protein expression, compared with the IR group. This result indicated that TST can reduce the number of myocardial apoptosis, which can nourish the myocardium, further to enhance the cardiac function of ischemia-reperfusion rats, and improve the hemodynamics in some degree.
Furthermore, AMPK, as an important protein kinase in the process of cell energy metabolism, can regulate Sirt1 activity and reduce the expression of in ammatory genes by activating AMPK, thus inhibiting the in ammatory response [44,45]. In addition, the reported study showed that TST can protect the acute liver injury by inhibiting NF-κB expression [46,47]. More importantly, it was reported that the activation of AMPK/Sirt1 could inhibited MIRI through alleviate in ammation reaction [32][33][34][35], which is very important for MIRI therapy in clinic. Thus, in this study, we further evaluated that whether TST plays an antiin ammatory role by stimulating AMPK/Sirt1/NF-κB signaling pathway [48]. The result showed that TST pre-treatment can increase the protein expression of p-AMPK and Sirt1, decrease the protein expression of acetylation NF-κB p65, compared with the IR group, which indicated that TST exerts the protective effect in MIRI rats may be through activation the signaling pathway of AMPK/Sirt1/NF-κB. Since the current study has many limitations, the study assessed only the effect of TST when it is pre-treated to the animals, the effectiveness of TST is to examine the long-term effects of TST treatment to cardiac function in rat models of IMRI or heart failure is a future direction for the work.

Conclusion
In summary, TST can inhibit in ammatory cell in ltration during MIRI, reduce the number of myocardial cell apoptosis, enhance cardiac function, and regulate the protein expression of p-AMPK and Sirt1, which further to improve hemodynamics, ensure blood perfusion and nutrition supply for the demand of surrounding tissues. The study will help to clarify the mechanism of MIRI and provide a new possible therapy method for the clinical patients.

Consent to publish
Not applicable.

Availability of data and materials
All data generated or analyzed during this study can be obtained from the corresponding Author with a reasonable request.

Competing Interests
The authors declare that they have no competing interests.

Funding
The study was funded by Beijing University of Chinese Medicine.
Authors' contributions WW and XZ designed and guaranteed the whole experiment studies; WW, JT, QX and DM carried out all the experiments; WW, JT, QX and DM analyzed the statistical data, WW drafted the manuscript; XZ edited and revised the whole manuscript; all authors read and approved the nal manuscript.     The protein expression of AMPK/Sirt1 signaling pathway in myocardium tissue of all group rats by western blot. A. The image showed a representative cropping blots. B. Quantitative analysis of the protein expression of p-AMPK, Sirt1 and Ac NF-κB p65. The image showed a representative cropping blots. ##P < 0.01 vs Sham group, *P < 0.05, **P < 0.01 vs IR group.