Progresses in the research of Danhong Injection against the cardial ischemia-referfusion injury

Zho Buchng（赵步长）， Zho To（赵涛）， Song Linlin（宋琳琳）*， Liu N（刘娜），Ji Yun'ning（贾苑凝）， Li Liuyun（李流云）， nd Ci Lingling（蔡玲玲）

a：Department of Cardiovascular Internal Medicine， Bu-Chang cardio-cerebrovascular Disease Hospital， Xi'an 710082， Chinab：Dermatology Department， DongFang Hospital of Beijing University of Chinese Medicine， Beijing 100078， China*Corresponding author： E-mail ：songlinlin577@sohu.com



Progresses in the research of Danhong Injection against the cardial ischemia-referfusion injury

Zhao Buchang（赵步长）a， Zhao Tao（赵涛）a， Song Linlin（宋琳琳）a*， Liu Na（刘娜）a，Jia Yuan'ning（贾苑凝）b， Li Liuyun（李流云）b， and Cai Lingling（蔡玲玲）b

a：Department of Cardiovascular Internal Medicine， Bu-Chang cardio-cerebrovascular Disease Hospital， Xi'an 710082， China
b：Dermatology Department， DongFang Hospital of Beijing University of Chinese Medicine， Beijing 100078， China
*Corresponding author： E-mail ：songlinlin577@sohu.com

ABSTRACT

Researches in the field of the myocardial ischemia-reperfusion injury are attracting the attentions of clinicians for the treatments that protect cardiac muscle cells from being injured can not only help the patients get recovery but also keep them in health. By clearing the free radicals and reducing calcium overload of myocardial cell， treatments with Danhong Injection will help myocardial cells survive from inflammatory reactions which are triggered by ischemia reperfusion so as that endothelial function will be improved and myocardial cell apoptosis will be inhibited. In all， Danhong Injection is an ideal medicine for protecting myocardial cell against ischemia reperfusion injury.

DanHong Injection； Myocardial ischemia reperfusion injury； Myocardial protection

Myocardial ischemia-reperfusion injury1（MIRI） is a normal pathologic process which always occurs in myocardial tissues when these tissues get the blood perfusion after blood flow being blocked in a short time. MIRI has lots of severe consequences such as reducing systolic function of myocardial cells， cutting coronary blood flow down and increasing vascular reaction， all of which will not only decrease the quality of life but also bring the poor prognosis to patients. So far， many medicines have been used to treat MIRI， such as nitrates， statins， calcium antagonists and angiotensin converting enzyme inhibitors （ACEI） and so on. However， long term of administration of these medicines will have side effects. So， clinicians are eager to looking for more ideal medicines with fewer side effects to treat MIRI. It has been appreciated that the treatment with Chinese Medicine has better outcomes with fewer side effects， which will provide prospects of finding some new options for the treatment of MIRI.

Danhong Injection， as a complex injection， is produced with modern pharmaceutical technology from a scientific prescription in which salvia miltiorrhiza and safflower are regarded as the main ingredients. Danhong Injection may play following functions such as promoting blood circulation， removing blood stasis and dredging meridian. Modern pharmacological researches have discovered that Danhong Injection can reduce the injuries generated by myocardial ischemia and platelet activation as well as slow formation of atherosclerotic plaque. Now， Danhong Injection is widely used for the treatment of cardio-cerebralvascular diseases， such as coronary heart disease， angina pectoris，and myocardial infarction. In all， Danhong Injection， which can restore the function of heart muscle cells in various ways， has some ideal effects on the protection of heart and brain against the injuries triggered by ischemia-reperfusion.

UNDERSTANDING OF MIRI IN TERMS OF TRADITIONAL CHINESE MEDICINE （TCM）2-4

There is no MIRI in the paradigm of TCM. Clinical symptoms of MIRI indicate that MIRI should be regarded as "palpitation" and "chest discomfort" with the Qi deficiency and blood stasis as pathological mechanisms while the blood stasis in heart is the etiology. So， the principles for the treatment of MIRI include improving blood circulation， removing blood stasis， tonifying Qi and dredging meridian as well.

Salvia miltiorrhiza and Safflower are the core ingredients in the prescription for producing DanHong Injection. In the paradigm of TCM， both ingredients can be used in removing blood stasis， relieving pain， improving the circulation of blood and dredging collaterals while the chemical analysis shows the differences between these two ingredients. From salvia miltiorrhiza， fat-soluble tanshinones， water-soluble phenolic acids as well as other effective ingredients can be extracted for the treatment of cardiovascular disease for these extracts can inhibit aggregation of platelet and lipid peroxidation of myocardial cells， reduce damages triggered by free radicals in the way of clearing them， maintain the stability of cell membrane，and enhance the tolerance of myocardial tissue to hypoxia. From safflower， safflower yellow pigments， catechins and other agents can be extracted， which can block the aggregation and activation of blood platelet， increase coronary blood flow and reduce the myocardial ischemia. In the prescription for producing DanHong injection， Salvia miltiorrhiza and safflower will play synergistic effects at the scientific ratio. So， DanHong Injection can also enhance the myocardial cell function and help these cells to avoid the ischemia-reperfusion injury in the ways of promoting blood circulation， clearing blood stasis as well as dredging meridian and collateral.

RESERCHES ON THE PROTECTIVE EFFECTS OF DANHONG INJECTION AGAINST MIRI

The role of DanHong Injection in clearing oxygen free radicals （OFRs）5-6

In the process of myocardial-ischemia reperfusion， a large number of OFRs will appear in the ischemia area， which will oxidize the unsaturated fatty acids and initiate the peroxide reaction in myocardial cell membrane. All of these reactions will cause some changes of cell membrane in structure and function. Reducing and clearing the OFRs are the methods to decrease the incidence of MIRI so as to protect the cardiac muscle cells. Some studies had indicated that DanHong Injection can help cells， which are in the area of ischemia reperfusion， to increase the production of endogenous antioxidant enzymes and endogenous superoxide dismutases （SOD）， to clear OFRs and to inhibit the peroxidation reaction in membrane so as to reduce the occurrence of MIRI and to help myocardial cells get recovery from the MIRI.

The role of DanHong Injection in reducing calcium overload5，6

Intracellular calcium overload plays a key role in myocardial reperfusion injury while abnormality of energy metabolism in myocardial can also trigger the occurrence of MIRI7. Under the condition of chronic myocardial ischemia， the distribution of calcium ion is out of order with the disturbance of Ca2+internal flow/separation although the total calcium concentration in myocardial remains unchanged. So， both in cytoplasm and mitochondria， the Ca2+concentration are higher than the normal level. When reperfusion occurs in local myocardial tissue， the Ca2+concentration in cytoplasm and mitochondria will continue to increase， which will lead to the interruption of producing ATP and myocardial spasm. And then， these cells will be injured and their function will be disturbed. In the case of ischemiareperfusion， the increase of intracellular Ca2+caused the calcium overload in myocardial cell. Researches had shown that Danhong Injection can effectively inhibit the interflow of Ca2+and lower Ca2+level in endochylema so as to retain the stability of cardiac muscle in function and structure as well.

The role of Danhong Injection in reducing inflammatory reaction in MIRI8，9

Inflammatory reaction， as a double-edged sword， always occurs in the whole process in cardiac ischemia-reperfusion. Despite the fact that it could clean the necrotic tissues， inflammatory reaction could harm the normal cells at the same time. Inflammatory agents such as IL-1， IL-6 and TNF-α played inevitable roles in the whole process. IL-1 is the trigger of inflammatory reaction. In the tissue with ischemia-reperfusion， IL-1 expression is higher than the normal level. IL-6 can induce neutrophiles to infiltrate toward ischemic tissue and surge these cells to phagocytose cardiac muscle cell. Other inflammatory factors such as TNF-α will increase adhesion of white blood cell in the ischemic area and bring the injury to endothelial cell. Furthermore，these inflammatory factors can also cause the high risk for the formation of blood clot. Researches had approved that Danhong Injection can degrade the level of IL-1， IL-6 TNF-α， decrease the adhesion of white blood cell and reduce inflammatory reaction.

The role of Danhong Injection in improving endothelial function

The initial step of MIRI is the dysfunction of endothelial cell，in which the synthesis and releasing of vasoactive agents are not as usual. So， the secretion of endothelium-derived relaxing factor（NO） begins to decrease or the activity of NO gets decreased while the releasing of ET-1 gets increased. These changes will cause the imbalance between dilators and constriction factors. It has been accepted that NO/ET-1 imbalance is a key factor in triggering and generating the ischemia reperfusion injury10，11. In the cardiac tissue which is undergoing the process of ischemic reperfusion process， NO fails to play its antagonistic effect against ET-1，which will increase in the reperfusion tissue. This will induce the strong vascular spasm/constriction and cause the serious ischemic and reperfusion injury to cardiac cells. Research12has shown that Danhong Injection can effectively inhibit the secretion of ET-1 and stimulate NO activity， recover the NO/ET balance so as to protect cardiac muscle from reperfusion injury.

The role of Danhong Injection in inhibiting cardiomyocyte apoptosis

When ischemic reperfusion is initiated， large amount of free radicals along with intracellular Ca2+overload and mitochondria abnormality will appear in local tissue， all of which willcontribute to the apoptosis of cardiac muscle cells. PI3K/Akt signaling pathway plays a crucial role against apoptosis， which attracts attentions of many researchers. Research had shown13that by activating PI3K/Akt signaling pathways， Danhong Injection can inhibit apoptosis of cardiac cell in the damaged tissue and prevent the occurrence of ischemic reperfusion injury in local tissue.

In summary， Danhong Injection could protect cardiac muscle tissue against the ischemic reperfusion injury in a variety of approaches.

THE CLINICAL OBSERVATION OF PROTECTIVE EFFECT OF DANHONG INJECTION AGAINST THE MIRI

Clinical researches indicated that patients would benefit from treatment with Danhong Injection. Zheng Xian-fu reported an 80-cases clinical observation14. All patients with coronary heart disease were diagnosed as heart blood stasis in the term of TCM and accepted percutaneous coronary intervention （PCI）. After PCI， eighty patients accepted the conventional therapy and then these patients were randomly divided into two groups. Forty cases accepted the treatment of Danhong Injection （the Danhong Injection 30mL + 0.9% normal saline 250mL by intravenous infusion） as treatment group while the rest cases entered the control group. 8-days was as a treatment cycle. Of all patients，troponin（TNI）， high-sensitivity C-reactive protein（hsCRP） and other biomarkers were measured at the time of admission while the same parameters were repeated one day as well as in one week after PCI respectively. Symptoms such as chest tightness and the occurrence of arrhythmias after PCI were also recorded in both groups. The results were encouraging. These patients，who accepted the treatment of Danhong Injection， improved their heat function with low-grade myocardial injury and inflammatory response as well as low incidence of reperfusion arrhythmia while the remission of chest pain was better than that of the control group. Furthermore， no case of adverse effects of Danhong Injection was reported in the treatment group.

Treatment with Danhong Injection is beneficial to the patients with myocardial infarction who accept thrombolytic therapy. Zhang Shan15collected sixty cases with myocardial infarction. After thrombolytic therapy， all patients with the diagnosis of Qi deficiency and blood stasis in the term of TCM， accepted the conventional therapy and then were divided randomly into two groups. 30 patients accepted the treatment of Danhong Injection，with 7d as a treatment cycle since the first day of thrombolytic therapy while the rests entered the control group. NT-pro-BNP，CK-MB， Hs-CRP and other biomarks were measured routinely while the remissions of patients' chest pain were recorded. The results confirmed that patients in the treatment group got benifits from the treatment of Danhong Injection with lower incidence of post-infarction angina， heart failure and reperfusion arrhythmias. The levels of NT-pro-BNP and Hs-CRP in the patients with the treatment of Danhong Injection were lower than that in control group. Furthermore， treatment of Danhong injection could help the patients relieve chest pain symptoms more effectively. All of these results had implied that Danhong Injection was an ideal medicine against MIRI.

Similar results were also observed by Han Guo-jie et al.16in the patient with acute myocardial infarction （AMI）. All 134 cases of AMI were randomly divided into two groups. 58 patients as control group received intravenous infusion of urokinase and took orally aspirin for thrombolysis. In two days after thrombolysis，they would receive subcutaneous injection of low molecular weight heparin for 5-7 days. Another 76 cases in the observation group not only received the treatment of Danhong Injection （20ml per day by intravenous infusion） on the basis of the treatment of the control group. for 14 days. Clinical data and the occurrence of complications would be documented for 4 weeks. The results show that although recanalization rate after thrombolysis were slightly higher in the observed group than that in the control group， the incidence of reperfusion arrhythmias， heart failure and post-infarction angina in the observed group was much lower than that in the control group while the rate of remission of chest pain was also higher in the observed group. These results indicated that Danhong Injection could be used as a protective agent against myocardial reperfusion injury.

A clinical observation conducted by Chen Hao et al17. convinced Danhong Injetcion could relieve the patient with acute myocardial infarction （AMI） in the way of resisting the reperfusion injury after PCI. 59 patients with AMI entered the research and were randomly divided into two groups while all of patients accepted the conventional treatment after PCI. 29 patients were selected as the treatment group and received the treatment of Danhong injection while 30 patients entered the control group. Endothelin （ET）， left ventricular end-diastolic volume， Left ventricular ejection fraction rate and ST segment drop rate in all patients were measured 1d， 7d， 14d after PCI respectively. The results had shown that ET in the treatment group reduced more significantly than that in the control group. Although the left ventricular end-diastolic volume increased onthe 7th day after PCI in both groups， the left ventricular enddiastolic volume would keep on increasing in control group on the 14th day while this parameter got reduced in the treatment group on the same day. Left ventricular ejection fraction rates in both groups were increased on the 7th day， the 14th day after PCI while on the 14th day， this parameter in treatment group increased more significantly than that in control group. On the 7th day after PCI， ST segment in the treatment group was significantly higher than that in the control group. These results showed that Danhong Injection would bring benefits to the patients in the ways of protecting endothelial cell， improving myocardial reperfusion and inhibiting myocardial remodeling of myocardial tissue.

DISCUSSION

Now， Danhong Injection is widely used for the treatment of cardiovascular and cerebrovascular diseases， especially for the treatment of ischemic heart disease and cerebrovascular disease. The main ingredients in Danhong Injection are tanshinones，salvianolic acids， safflower yellow and so on， which can be used as anticoagulant agent for myocardial protection. These ingredients can not only improve microcirculation， reduce platelet adhesion and aggregation， but also increase the activity of plasmin for fibrinolysis.

By clearing oxygen free radicals， reducing lipid peroxidation and intracellular calcium overload， Danhong Injection will restrain inflammatory reaction so as to resist the MIRI. As a protective agent， Danhong Injection can inhibit apoptosis and improve myocardial energy metabolism at the occurrence of MIRI. Furthermore， clinical researches have confirmed that Danhong Injection， as a protectective agent against myocardial ischemia-reperfusion injury， can protect vascular endothelium and reduce the incidence of complications induced by MIRI.

In all， Danhong Injection is an ideal medicine to alleviate MIRI while clinical observations have convinced that treatment with Danhong Injection could bring many encouraging benefits to patients. So， the wide utilization of Danhong Injection will provide new options for the patients with cardio cerebrovascular diseases.

REFERENCES

1 Schulze CJ， Wang W， Kumari R， et a1. Imbalance between tissue inhibitor of metalloproteinase-4 and matrix metallopreteinases during acute myocardial correction of myocardial ischemiareperfusion injury. J Circulation， 2003， 107 （19）： 2487-2492.

2 Liang LC， Zhang SC.TCM differentiation of MIRI. China Medicinal Herald，2009，6（24）：9-12.

3 Tang DL， Liu ZH， Zhang HM. A modern studied review of TCM pathogenesis of MIRI and clincial treatment. China Journal of Basic Medicine in Traditional Chinese Medicine，2011， 17（9）：1051-1052.

4 Wu P.A review of treating CVD with the Danhong injection. Guide of China Medicine， 2014， 12（6）：41-42.

5 Liu SZ， Yang SQ.A study review of pathogenesis MIRI. Practical Journal of Clinical Medicine， 2007， 4（1）：88-90.

6 Ji HG， Si L.A study review of protecting MIRI with Danshen. Guangming Journal of Chineas Medicine， 2006，21（3）：52-54.

7 Lu JP， Jing L.Protective Effect of Breviscapine on Myocardial Ischemia Reperfusion Injury in Rabbits. Journal of Tradintonal Chinese Medicine， 2010， 51（11）：1031-1037.

8 Pchejetski D， Kanduzova O， DayonA， et al. Oxidative stressdependent sphingosine kinase-1 inhibition mediates monoamine oxidase A-associated cardiac cell apoptosis. Circ Res， 2007，100（1）：7-9.

9 Wang J， Liang Z， Huo Y， et al.A study of efficacy of the Danhong injection on blood serum adhesion molecule 1 and inflammatory factor in MIRI rats.Liaoning Journal of Traditional Chinese Medicine， 2012，39（5）：782-784.

10 Xu SY， Bian RL， Chen X. Methodology of Pharmacological Experiment. Beijing： People's Medical Publishing House ，2002：1834.

11 Zhang DF.Experiments of pharmacology of TCM and pharmacology. Shanghai： Shanghai science and Technology Press ， 2002：8.

12 Gong YB， Qi YB， Qi GX， Li G. Efficacy of the Danhong injection on serum nitric oxide synthase and endothelin-1 in MIRI rats.China Foreign Medical Treatment， 2007（23）：76.

13 Wang P， Zhang YD.Effect of danhong iniection on cell apoptosis in rats with myocardial ischemia-reperfusion injury. Jiangsu Medical Journal， 2010， 38（2）：143-145.

14 Zheng XF.Clinical observation on treating MIRI following PCI in CHD patients of the Xinxue Yuzu type. Fuzhou： Fujian University of Traditional Chinese Medicine，2012，

15 Zhang GS. Clinical observation on treating reperfusion injury after TIMI of the Qixu Xueyu type.Fuzhou： Fujian University of Traditional Chinese Medicine，2012.

16 Han GJ， Liu WH， Yang DW.Treating reperfusion injury in acute myocardial infarction with the Danhong injection. People's Military Surgeon ， 2012， 55（6）：514-515.

17 Chen H， Zhao LX， Chu RPet a1. Protective effects of the Danshen injection on myocardium following AMI intervention. Hebei Medical Journal， 2010， 32（11）：1391-1392