DG has been reported to have a wide range of pharmacological properties, such as vasodilation, anti-atherosclerosis and an anti-oxidative effect
[29–33, 37, 38]. Most particularly, DG pretreatment induced cardioprotection against an I/R injury in rats by enhancing the mitochondrial antioxidant status by activating PKCε to inhibit the opening of KATP in mitochondria
[31, 32]. Our present study aims to explore whether the administering of DG post-treatment in the reperfusion/reoxygenation phase can directly provide protective effects against reperfusion injury ex vivo and in vitro. It was found that DG could protect the rat heart damage in the reperfusion phase by inhibiting the release of heart specific enzymes and restoring the contractile force as well as coronary flow rate recovery. In addition, DG directly and rapidly attenuated intracellular Ca2+ accumulation within H9c2 cells in the reoxygenation phase. DG also could inhibit the apoptosis of H9c2 cells. Our results provided evidence that there is a potential for patients with coronary heart diseases to attenuate the I/R injury in their blood restore surgeries by DG.
In our previous study, the contents of seven components, namely danshensu, protocatechuic aldehyde, puerarin, daidzein 8-capiosyl-glucoside, daidzin, salvianolic acid B and daidzein were quantified in DG water extract. Since only water extract was used, the valuable lipophilic tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) were not present in the DG extract or only in traces. Therefore, tanshinones did not contribute but water soluble marker such as salvianolic acid B might contribute to the cardiovascular protective activities
In the ex vivo study, the recovery of coronary flow rate and of the contractile force in the reperfusion phase was examined. Oxygen delivery in the heart is dependent on two crucial factors, namely, the blood flow rate and the arterial oxygen content
. Flow rate recovery could be helpful for restoring oxygen and nutrient supply and in the recovery of the heart function. In the present study, DG post-treatment obviously improved the coronary flow rate and contractile force recovery after an I/R challenge in a dose-dependent manner. In the high dosage DG treatment group, the contractile force recovery of the damaged heart was up to 60% (Figure
1A) and the coronary flow rate was almost totally recovered (Figure
1B). DG treatment significantly inhibited CK and LDH release in the hearts (Figure
1D). It reflected that heart damage status had been partly reversed and was consistent with the results of contractile force and coronary flow rate recovery.
An I/R challenge always results in cardiac dysfunction and cellular injury through inducing a calcium overload and destroying the contractile apparatus (Troponin I, a membrane skeleton protein)
[41, 42]. In the cellular model, it was found that membrane integrity of cardiomyocytes was protected by DG decoction by improving the expression of Troponin I (Figure
3). It underlines that DG post-treatment could restore the heart contraction function after an I/R challenge, by protecting the cell structure and the contractile unit integrity of cardiomyocytes. Additionally, DG treatment improved the survival of H9c2 cells after a H/R challenge (Figure
4 showed that the number of early apoptotic cells was obviously decreased after DG treatment. A high concentration of DG also significantly inhibited the late apoptosis of H9c2 cells. Inhibition of apoptosis was also examined by measuring the expression of pro-apoptosis and anti-apoptosis protein. The result showed that the expression of caspase 3 (pro-apoptotic factor) was significantly decreased and bcl 2 (anti-apoptotic factor) was significantly increased with DG treatment (Figure
5A). The increased survival ability of cardiomyocytes could improve the heart function recovery after an I/R challenge.
Over recent years, mitochondria have become a major research focus for experimental cardiologists. The exploration of the potential role of mitochondria in the pathogenesis of cardiovascular diseases, particularly in an I/R injury, has been greatly increased
[43–45]. The opening of mPTP in the outer-membrane of mitochondria is a key pathological phenomenon involving in an I/R injury
. One of its downstream events is the rupture of the outer mitochondrial membrane, which could become permeable to molecules smaller than 1.5 kDa, and which then would induce the release of cytochrome c
. It is well known that when cyotochrome c is released from mitochondria, this could couple with Apaf-1, forming a macromolecular complex which recruits and activates the death effector caspase 9
. The activated caspase 9 then activates caspase 3 and caspase 7, which in turn activates the early apoptosis process
. To further investigate the molecular mechanism of anti-apoptotic activity of DG, the nature of the cytochrome c release after a H/R challenge was determined. Figure
5B showed that DG suppressed the release of cytochrome c from mitochondria to cytosol by comparing its expression between cytosol and mitochondria. The other event is the opening mPTP which causes mitochondria to become depolarized and loses its Δψ. This event is involved in early apoptosis and further induce the proton and other molecules going out of the outer mitochondrial membrane
[44, 46]. Figure
6 showed that DG suppressed the mitochondrial depolarization of H9c2 in a dose-dependent manner. The results implied that DG could provide anti-apoptotic protection to the cardiomyocytes through inhibiting the opening of mPTP induced by a H/R injury. This is consistent with the previous report that pretreatment of DG inhibited the opening of MPT pores on rat hearts with an I/R challenge
Calcium ion stunning also plays a critical role in the initiation of apoptosis in cardiomyocyte in an I/R injury
. Our present results revealed that DG treatment greatly inhibited a H/R-induced elevation of cytosolic calcium ion level through living cell recording system under the confocal microscope (Figure
7). The results suggested that the protection by DG may be mediated partly by decreasing the calcium influx into H9c2 cells. As a result, DG may improve the heart function recovery in an I/R Langendorff model by inhibiting the calcium overload within cardiomyocytes. Under an I/R or H/R situation, an ATP lack could induce the sodium overload caused by inhibition of the sarcolemmal Na+-K+-ATPase and calcium pump
[51, 52]. The sodium overload would cause calcium overload via increased Na+-Ca2+ exchange in the reperfusion injury
. This revealed the fact that DG may suppress the calcium overload to improve the heart function recovery by protecting the activity of Na+-K+-ATPase, calcium pump and Na+-Ca2+ exchanger. Our present result is consistent with a previous report that water extract of Danshen attenuated anoxia and reoxygenation-induced [Ca2+
i increase in rat cardiac ventricular myocytes and that DG pretreatment decreased the calcium concentration in the rat heart tissue with an I/R injury
A limitation of this study is the use of ex vivo model instead of in vivo model. The reason is that the animal model of myocardial infarction (MI) which is mimicked by Langendorff heart model, is facing the high mortality rate of the surgical procedure. In brief, after anaesthesia, orotracheal intubation and thoracotomy, the heart is rapidly exteriorised and the coronary artery is ligated in the proximal segment using a thin thread. The occlusion of the artery can be recognised by blanching of the tissue distal to the ligation. In this complicated surgical procedures, a comparative study showed that the mortality of MI in Sprague–Dawley rats was 36%
. Therefore, ex vivo Langendorff heart model was used in this study instead of in vivo model.