The Protective Effect Of Cistanche Total Glycosides On The Myocardial Toxicity Of Mice Induced By Adriamycin

For more information:ali.ma@wecistanche.com

Yulia Yiming'

[Project supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region Science and Technology Commission (96814)]

[Profile of the author, Eliya Yiming (1964-), female (Uyghur), master. Lecturer, Research direction: Cardiovascular pharmacology. ],

Wang Xiaowen', Wang Xuefei', Heiliman Yilahong 2, Xie Yaoyun 2, Du Niansheng 3

(Xinjiang Medical University|Department of Pharmacology, School of Pharmacy, Department of Electron Microscopy, School of Basic Medical Sciences, Department of Chemistry of Natural Medicines, School of Pharmacy, Urumqi 830054, Xinjiang)

Abstract: To explore the protective effect and mechanism of Cistanche glycosides (GCs) on doxorubicin (Dox)-induced myocardial damage in mice. Fang Xie: A model of acute myocardial injury caused by intraperitoneal injection of Dox (17.5 mg/kg) in NIH mice was used to determine the activity of myocardial superoxide dismutase (SOD); selenium-glutathione peroxidase (Se-GSH- Px) activity; malondialdehyde (MDA) contains serum creatine phosphokinase (CPK) activity. Electron microscopy examines the ultrastructural changes of cardiomyocytes. Results: 48 hours after intraperitoneal injection of Dox can cause significant damage to the myocardium of mice. Myocardial SOD and Se-GSH-Px activity decreased, MDA content increased, serum CPK activity increased, and myocardial tissue ultrastructure damage occurred. Cistanche glycosides (62.5, 125.0, 250.0 mg/kg) can increase myocardial SOD, Se-GSH-Px activity. Reduce MDA content and reduce CPK release. Reduce myocardial ultrastructure damage. Jie Leng: Cistanche total glycosides have a certain protective effect on myocardial damage caused by doxorubicin. Its mechanism may be related to its protection of myocardial SOD and Se-GSH-Px activity, scavenging of free radicals, and preventing lipid peroxidation.

Keywords: doxorubicin; total glycoside of cistanche; cardiotoxicity; lipid peroxidation; creatine kinase; ultrastructure

Doxorubicin (Dox) is a kind of onion antibiotic, which has a good effect on a variety of malignant tumors, but due to its acute and chronic myocardial toxicity, it limits the wide application of Dox in clinical practice. It is known that the anti-tumor effect of Dox is to affect DNA replication and RNA synthesis, and its cardiotoxicity is related to the lipid peroxidation damage induced by semi-adriamycin. Therefore, it is important to find free radical scavengers and antioxidants to antagonize the cardiotoxicity of Dox while retaining its anti-tumor activity. The total extracts of Cistancheare the active ingredients extracted from Cistanche. Relevant studies have found that Cistanche glycosides have antioxidant effects on mouse tissues, Cistanche glycosides can significantly reduce lipofuscin content (3), Cistanche glycosides have protective effects on rat myocardial ischemia (3), and Cistanche glycosides have anti-lipid peroxidation and anti-radiation effects. To explore the protective effect and mechanism of Cistanche glycosides on Dox-induced myocardial injury in mice.

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1 Material and methods

1.1 Drugs and Reagents

Cistanche glycosides are extracted from Cistanche herb in northern Xinjiang, and the main ingredients are echinacea, ergot ethoxylates, etc. (provided by the Department of Natural Medicine Chemistry, School of Pharmacy, Xinjiang Medical University); tetra methoxy propane (TMP Sigma Company); Adriamycin (Dox, Zhejiang Hisun Pharmaceutical Co., Ltd.); Vitamin E (Vit E, Shanghai Yan'an Pharmaceutical Factory); other reagents are domestically produced.

1.2 Experimental equipment

721 type spectrophotometer (Shanghai No. 3 Analytical Instrument Factory); OLYMPUS microscope (made in Japan); JEM-100CXII transmission electron microscope (made in Japan)? PHS-250 type acidity meter (Shanghai Lei Magnetic Instrument Factory).

1.3 Animal grouping and administration

NIH mice are provided by the Animal Experimental Center of Xinjiang Institute of Endemic Diseases. The medical laboratory animal certificate number is No. 16-068. 138 NIH mice were selected, weighing (24±2) g, half male and half female. Randomly divided into 6 groups (23 in each group): (1) control group: normal saline (NS) 20 ml/kg; (2) Dox injury group: NS 20 ml/kg; (3) Vit E group: Vit E 100 mg /kg; (4) GCs i group: GCs 62.5 mg/kg; (5) GCs i group: GCs 125.0 mg/kg; (6) GCS1 group: GCs 25.0 mg/kg, each of the above groups All were given by intragastric administration, once a day. Dox injury group, Vit E group, GCs | group, GCs I group, GCs I group were intraperitoneally injected with Dox 17. 5 mg/kg on the 4th day after NS and GCs were injected for 48 hours, the eyeballs were taken and blood was taken, serum was prepared, and immediately The animals were sacrificed to take out the heart, rinsed with NS, blotted dry with filter paper, and weighed. Underwater bath conditions, the myocardial homogenate was prepared with NS, centrifuged at 3 500 r/min, 30 min, and the supernatant was taken to measure biochemical indicators. There were 3 mice in each group, and the hearts were taken to make electron microscope specimens.

1.4 Determination of biochemical indicators

The pyrogallol autoxidation method was used to determine the myocardial superoxide dismutase (SOD) activity; the DTNB method was used to determine the myocardial selenium-glutathione peroxidase (Se-GSH-Px) activity Use the TBA method to determine the content of myocardial lipid peroxidation product malondialdehyde (MDA); protein quantification uses the CBB-SDS method called the CPK method to determine the serum creatine phosphokinase (CPK) activity.

1.5 Electron microscopy of myocardial tissue

The apex of the heart was fixed with 4% glutaraldehyde and 1% acid, dehydrated with acetone, embedded in Epon 812, and observed under the transmission electron microscope with lead-uranium electron staining JEM-100CX I.

1.6 Statistical processing

All data are represented by Wang Shi s. After the experimental data is tested for homogeneity of variance, the F test and q test are used for statistical processing. Inspection level a = 0.05.

2 Results

2.1 The effect of Cistanche glycosides on the biochemical indexes of Dox-injured myocardium is shown in Table 1.

2. 1- 1 The effect of Cistanche glycosides on the SOD activity of Dox injured myocardium.

Compared with the control group, the Dox injury group reduced the myocardial SOD activity by 41.1% (P V0.01). Compared with the Dox injury group, it increased by 30.9%, 33.2%, 36.5%, and 34.9% (all P<0.01), the GCS1 group was close to the control group (P>0.05), and the remaining groups were all lower than the control group.

2.1.2 Effect of GCs on Dox-injured myocardial Se-GSH-Px activity.

Compared with the control group, the Dox injury group has a 26.2% reduction in myocardial Se-GSH-Px activity (P<0.01), GCs, GCs, Compared with the Dox injury group, the GCs, group, and Vit E group increased by 19.2%, 21.1%, 25.0%, and 26.1% respectively (F<0.01), which were all close to the control group (P>0 . 05).

2.1.3 The effect of Cistanche glycosides on the MDA content of the Dox-injured myocardium was reduced by 16.7%, 17.3%, 18.6%, and 17.3% respectively compared with the Dox-injured group (Fall V0.01), which were all close to the Control group (P>0.05).

2.1.4 The effect of GCs on serum CPK activity in Dox injured mice.

Serum CPK activity in the Dox injury group increased by 87.3% ( <0.01), Cistanche glycosides I, Cistanche glycosides group, and Vit Compared with the Dox injury group, the E group decreased by 26. 6%, 33.1%, 37.4% and 36. 7% (all F<0.01), which were higher than those of the control group.

2.2 Electron microscopy of mouse myocardium

2.2.1 In the control group, the myocardial fiber sarcoma was intact, the myofibrils were arranged in an orderly manner, the sarcomere bands were clearly structured, the mitochondria were neatly arranged, the structure was normal, and the nuclear structure was normal.

2.2.2 Dox injury group

It can be seen that there are obvious changes in the ultrastructure of myocardial fibers, myocardial cells are swollen, the intracytoplasmic sarcomere grows, the bright band widens, abnormal contraction bands appear in some areas, and the Z-line material increases. Mitochondria were diffusely swollen, hyperplastic, sparsely arranged, broken, and vacuolated. The sarcoplasmic reticulum expands, secondary lysosomes increase, and focal degeneration can be seen. The volume of the nucleus increases and the perinuclear space widens.

2. 2.3 The swelling of myocardial fibers in the Cistanche glycosides I group was reduced.

The sarcomere structure is basically normal, but the sarcomere is still wider than the control group. The swelling of mitochondria has been reduced, the density of the matrix has been increased, the density of the mitochondria has increased, and individual mitochondria still have changed. Increased secondary lysosomes in the cytoplasm.

2.2.4 The myocardial fiber structure of the Cistanche glycosides I, I group basically recovered to normal.

Myofibrils are arranged neatly, the structure of the sarcomere is normal, and the structure of mitochondria returns to normal.

3 Discussion

Dox is a kind of onion antibiotic, which has the characteristics of a broad anti-tumor spectrum and strong effect. However, Dox can cause severe myocardial toxicity, such as various arrhythmias in the early stage of medication, dose-dependent, and congestive heart failure in the late stage, which limits its clinical application. The main mechanism of Dox causing myocardial toxicity is the production of excessive reactive oxygen free radicals that cause myocardial damage. The affinity of Dox with myocardial tissue is significantly higher than that of other tissues. After entering myocardial cells, Dox transforms into semi-dox, which acts on oxygen molecules to transform them into superoxide anion free radicals (Oxid), accompanied by the production of hydrogen peroxide (H2O2). The MDA content of Ol and H2O2 in the Haber-Weiss Dox injury group increased by 25.8% compared with the control group (P <0.01), and the Cistanche glycosides |, Cistanche glycosides 1, Cistanche glycosides group, and Vit E group were reduced by 16.7% compared with the Dox injury group. , 17. 3%, 18. 6% and 17. 3% (Fall V0.01), which are all close to the control group (P>0.05). The reaction or Fenton reaction produces hydroxyl radicals (OH • ). 0$ and OH • A large amount of accumulation can cause lipid peroxidation in tissues and cell membranes and cause damage to biological macromolecules.

In the experiment, it was observed that after 48 hours of intraperitoneal injection of Dox (17.5 mg/kg) to mice, the activities of SOD and Se-GSH-Px in the mouse myocardium were significantly reduced, and the content of lipid peroxidation product MDA was significantly increased. High, serum CPK activity increases. Under the electron microscope, the ultrastructure of the heart muscle was damaged, manifested as obvious swelling of mitochondria, rupture of mitochondria, vacuole degeneration, reduction of matrix density, and expansion of sarcoplasmic reticulum.

All dose groups of GCs can significantly increase the activities of free radical scavenging enzymes SOD and Se-GSH-Px in the heart muscle of Dox-injured mice, reduce the content of MDA and reduce the release of CPK. It is suggested that Cistanche glycosides can reduce the lipid peroxidation damage induced by Dox by increasing the activity of free radical scavenging enzymes in the body. Cistanche glycosides, groups significantly protect the ultrastructure of myocardial fibers from Dox damage. Detected in vitro with modern chemiluminescence analysis technology and found that Cistanche glycosides can be effectively eliminated. Only active oxygen radicals such as OH • and H2O2 are correct. The scavenging effect of a ganglion is particularly significant "⑵. It is suggested that Cistanche glycosides can clear the OR induced by semi-dox in the myocardium, thereby interrupting the chain reaction of free radicals and acting as a blocker of the lipid peroxidation chain.

In this experiment, Vit E was used as a positive control. The results showed that Vit E has a certain protective effect on Dox-induced acute myocardial damage in mice. It can reduce myocardial MDA content and increase SOD and Se-GSH-Px activities. According to the literature [13], The reports are consistent.

The main component of Cistanche glycosides is phenethylamine, and its structure is similar to Vit E, that is, it has a phenolic hydroxyl group and a hydroxyl group. Therefore, it is speculated that Cistanche glycosides can provide the hydrogen atoms on the phenolic hydroxyl group of its molecular structure to the lipid radical (LOO -), which turns it into lipo hydroperoxide (LOOH), which is then decomposed into non-toxic hydroxyl by GSH-Px. It prevents the peroxidation of unsaturated fatty acids in the biofilm by free radicals, thereby reducing the damage of Dox to cardiomyocytes. The mechanism remains to be studied in depth.

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