Whether Glycosides Of Cistanche Can Be Used To Treat Cardiotoxicity?

The protective effect of glycosides of cistanche against doxorubicin-induced cardiotoxicity in mice

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Wu Liya, Wang Xiaowen, Wang Xuefei, et al

( Department of Pharmacology, X Xinjiang Medical University, X Xinjiang 830054)

Abstract: Objective: To investigate the protective effect of glycosides of cistanche ( GCs) against doxorubicin-induced cardiotoxicity in mice and its mechanism.

Methods: NI H mice were treated intraperitoneally with doxorubicin ( Dox ) at a single dose of 17. 5 mg /kg- 1 to develop an acute myocardium injury model. The activity of SOD, GS H-Px, the content of M DA, and the activity of creatine phosphokinase ( CPK) were measured. The cardiac ultrastructural changes were examined with a scanning electron microscope.

Results: At the 48h after administration, Dox elicited severe myocardial damage with decreasing my oca radial SOD and GS H-Px activity, increasing myocardial M DA content and serum CPK activity in mice. It also caused severe myocardial cell damage at the ultrastructure level. Oral administration of GCs ( 62. 5, 125, 250 mg /kg- 1 ) protected against these changes induced by Dox, by increasing myocardial SOD, GS H-Px activity, decreasing myocardial M DA content, and serum CPK activity, and reducing cardiotoxicity o Dox at the ultrastructural level.

Conclusion: The glycosides of Cistanche have a protective effect on cardiotoxicity induced by Dox. The mechanisms of reduced cardiotoxicity induced by Dox may depend on the effect of GCs on scavenging ox yg en free radicals in mice hearts, protecting the activity of SO D and GSH-Px, and inhibiting lipid peroxidation.

Keywords: doxorubicin; glycosides of cistanche; lipid peroxidation; cardiotoxicity; creatine phosphokinase; ultrastructure

Doxorubicin (Dox) is an anthraquinone antibiotic and has a good effect on a variety of malignant tumors. However, 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 affects DN A replication and RN A synthesis, and its cardiotoxicity is related to the lipid peroxidation damage induced by semiquinone adriamycin [1, 2]. Therefore, it is important to find free radical scavengers and antioxidants to antagonize the cardiotoxicity of Dox while retaining its anti-tumor activity. Cistanche cistanche total glycosides (GCs) are active ingredients extracted from Cistanche. Relevant studies have found that GCs have antioxidant effects on mouse tissues, can significantly reduce lipofuscin content [3], have protective effects on rat myocardial ischemia [4], and have anti-lipid peroxidation and anti-radiation effects. [5]. This study aims to explore the protective effect and mechanism of GCs on myocardial injury in mice induced by Dox.

1 Material and methods

1. 1 Medicine and reagents

GCs are extracted from the salt-producing Cistanche in northern Xinjiang. The main ingredients are phenethyl alcohol glycosides such as echinacoside and ergot glycosides (provided by the Department of Natural Medicine Chemistry, School of Pharmacy, Xinjiang Medical University); Tetramethoxypropane (TM P Sigma Company) ); Doxorubicin (Do x, Zhejiang Hisun Pharmaceutical Co., Ltd.); Vitamin E (Vit E, Shanghai Yan'an Pharmaceutical Factory); other reagents are domestically produced.

glycosides of cistanche's effect on cardiotoxicity

1. 2 Experimental equipment

721 type spectrophotometer (Shanghai Third Analytical Instrument Factory); O LYM PU S 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

N IH mice were provided by the Animal Experiment Center of Xinjiang Institute of Endemic Diseases, and the medical laboratory animal certificate number: No. 16-068. 138 N IH mice were selected, weighing (24±2) g, half male and 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: Vito 100 mg /kg; (4) GCsⅠ group: GCs 62.5 mg/kg; (5) GCsⅡ group: GCs 125.0 mg/kg; (6) GCsⅢ group: GCs 25.0 mg/kg, all of the above groups were irrigated Stomach administration, once a day. Dox injury group, Vit E group, GCsⅠ group, GCsⅡ group, GCsⅢ group were intraperitoneally injected with Dox 17. 5 mg/kg on the 4th day after administration of NS and GCs. After 48 hours, the eyeballs were removed to collect blood, serum was prepared, and the animals were immediately sacrificed to take out the heart, rinsed with NS, blotted dry with filter paper, and weighed. After 30 minutes, take the supernatant to measure the biochemical indicators. There were 3 mice in each group, and the hearts were taken to make electron microscopy specimens.

1.4 Determination of biochemical indicators

The pyrogallol autooxidation method [6] was used to determine the activity of myocardial superoxide dismutase (SOD); the DTNB method [7] was used to determine the activity of myocardial selenium-glutathione peroxidase (Se-GS H-Px); Using TBA method [8] to determine the content of myocardial lipid peroxidation product malondialdehyde (M DA ); protein quantification using CBB-SDS method [9 ]; using CPK method [10] to determine serum creatine phosphokinase (CPK) activity.

1.5 Electron microscopy of myocardial tissue

The apex of the heart was double fixed with 4% glutaraldehyde and 1% osmium acid, dehydrated with acetone, embedded in Epon 812, stained with lead-uranium electrons, and observed under a JEM-100CXII transmission electron microscope. 1.6 Statistical processing All data are expressed as x-± s. After the experimental data is tested for homogeneity of variance, the F test and q test are used for statistical processing. Inspection level α = 0.05.

2 results

2.1 The influence of GCs on the biochemical indexes of Dox-injured myocardium is shown in Table 1.

2. 1.1 The effect of GCs on SOD activity of Dox-injured myocardium

Compared with the control group, the Dox injury group decreased myocardial SOD activity by 41.1% (P <0.01). The GCsⅠ, GCsⅡ, GCsⅢ, and Vit E groups increased the myocardial SOD activity by 30.9% and 33.2 compared with the Dox injury group, respectively. %, 36.5%, and 34.9% (all P<0.01), the GCsⅢ group was close to the control group (P>0.05), and the rest of the groups were lower than the control group.

2.1.2 The effect of GCs on the activity of Se-GSH-Px in the myocardium damaged by Dox

Compared with the control group, the Dox injury group decreased the myocardial Se-GSH-Px activity by 26.2% (P <0.01), and the GCsⅠ, GCsⅡ, GCsⅢ, and Vit E groups increased by 19. 2% and 21 respectively compared with the Dox injury group. .1%, 25.0% and 26.1% (all P<0.01), which are all close to the control group (P>0.05).

2.1.3 The influence of GCs on the content of M DA in Dox-injured myocardium

Compared with the control group, the MDA content of the Dox injury group increased by 25.8% (P <0.01), and the GCsⅠ, GCsⅡ, GCsⅢ, and Vit E groups decreased by 16.7%, 17.3%, and 18. 6% and 17.3% (both P<0.01), both were close to the control group (P>0.05).

glycosides of cistanche

2.1.4 The effect of GCs on the activity of serum C PK in Dox-injured mice

Serum CPK activity in the Dox injury group increased by 87.3% compared with the normal group (P <0.01), and the GCsⅠ, GCsⅡ, GCsⅢ, and Vit E groups were reduced by 26.6%, 33.1%, and 37 respectively compared with the Dox injury group. . 4% and 36. 7% (both P<0.01), both higher than the control group.

Table 1 The effect of GCs on the biochemical indexes of Dox injured myocardium

Note: Compared with the control group, * * P <0.01; compared with the Dox injury group, # # P <0.01

2.2 Electron microscopy of mouse myocardium

2. 2. 1 Control group

The myocardial fiber membrane is intact, the myofibrils are arranged in an orderly manner, the sarcomere bands are structured, the mitochondria are arranged neatly, the structure is normal, and the nuclear structure is normal.

2. 2. 2 Dox damage 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 and hyperplastic, with sparse cristae arrangement, fractures, and vacuolar degeneration. The sarcoplasmic reticulum expands, secondary lysosomes increase, and focal degeneration can be seen. The nuclear volume increases and the perinuclear space widens.

2. 2. 3 GCsⅠ group

Myocardial fiber swelling has been reduced. The sarcomere structure was normal, but the sarcomere was still wider than the control group. Mitochondrial swelling was reduced, matrix density increased, cristae density increased, and individual mitochondrial cristae still changed. Increased secondary lysosomes in the cytoplasm.

2. 2. 4 GCs Ⅱ and Ⅲ groups

The myocardial fiber structure returned to normal. The myofibrils are arranged neatly, the sarcomere structure is normal, and the mitochondrial structure returns to normal.

3 Discussion

Dox is an anthraquinone antibiotic, which has the characteristics of a broad anti-tumor spectrum and strong effects. However, Dox can cause serious 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 mechanism of Dox causing myocardial toxicity is mainly 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, it is converted into semiquinone Dox, which acts on oxygen molecules to convert it into superoxide anion free radicals (O-2), and is accompanied by hydrogen peroxide (H2 O2) )generate. O-2· and H2 O2 generate hydroxyl radicals (OH·) through Haber-Weiss reaction or Fento n reaction. O-2· and O H· accumulate in large quantities, which can cause lipid peroxidation in tissues and cell membranes and cause damage to biological macromolecules [11].

It was observed in the experiment that after 48 hours of intraperitoneal injection of Dox (17.5 mg/kg) to mice, the activities of the mouse myocardial free radical scavenging enzyme SO D and Se-GSH-Px were significantly reduced, and lipid peroxidation products The content of MDA increased significantly, and the activity of serum CPK increased. Under the electron microscope, the ultrastructure of the cardiac muscle was damaged, manifested as obvious swelling of mitochondria, fractured cristae, vacuolar degeneration, reduced matrix density, and expansion of sarcoplasmic reticulum.

All dose groups of GCs can significantly increase the activities of the free radical scavenging enzymes SOD and Se-GS H-Px in the heart muscle of Dox-injured mice, reduce the content of MDA, and reduce the release of CPK. It is suggested that GCs can reduce the lipid peroxidation damage induced by Do x by increasing the activity of free radical scavenging enzymes in the body. The GCsⅡ and GCsⅢ groups significantly protected the ultrastructure of myocardial fibers from Dox damage. In vitro detection using modern chemiluminescence analysis technology, it is found that GCs can effectively remove active oxygen free radicals such as O·2, O H·, H2O2, and the scavenging effect on O·2 is particularly significant [12]. It is suggested that GCs can scavenge the O-2· induced by semiquinone 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 acute myocardial damage caused by Do x in mice. It can reduce the content of myocardial M DA and increase the activities of SOD and Se-GSH-Px. 13] The reports are consistent.

The main component of GCs is phenoxyethanol glycosides, and the structure is similar to that of Vit E, that is, it has a phenolic hydroxyl group and a hydroxyl group. Therefore, it is speculated that GCs can provide the hydrogen atoms on the phenolic hydroxyl group of its molecular structure to the lipid radical (LOO·), turning it into lipohydroperoxide (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.

glycosides of cistanche can treat cardiotoxicity

References

[1] Baser RL, Green M D. Complication of treatment: strategies for prevention of a recycling e cardiotoxicity [J]. Cancer Tread Rer, 1993, 19: 57.

[2] Doroshow JH. Eff ect of anthracycline antibiotics on ox yg en radical formation in rat heard [J]. Cancer Res, 1983, 43: 460.

[3] Wang Xiaowen, Li Linlin, Muhu Yati, et al. Antioxidant effect of total glycosides of Cistanche on mouse tissues [J]. Chinese Journal of Chinese Materia Medica, 1998, 23(9): 554-556.

[4] Mao Xinming, Wang Xiaowen, Li Linlin, et al. Protective effect of total glycosides of Cistanche on myocardial ischemia in rats [J]. Chinese Herbal Medicine, 1999, 30(2): 118-120.

[5] Li Linlin, Wang Xiaowen, Wang Xuefei, et al. Anti-lipid peroxidation and anti-radiation effects of total glycosides of Cistanche [J]. Chinese Journal of Chinese Materia Medica, 1997, 22(6): 364-367.

[6] Zou Guolin, Gui Xingfen, Zhong Xiaoling, et al. A SOD determination method-improvement of pyrogallol autoxidation method [J]. Progress in Biochemistry and Biophysics, 1986, 4: 17.

[7] Xia Yiming, Zhu Lianzhen. Determination of glutathione peroxidase activity in blood and tissues-DTNB direct method [J]. Hygiene Research, 1987, 16(4): 29-32.

[8] Chen Shunzhi, Jin Youyu. Comparison of three methods of lipid peroxide TB A color development [J]. Journal of Clinical Laboratory Science, 1984, 2(4): 8-10.

[9] Macart M, Gerbaut L. An improv em ent of the coomassie blue dye-binding method cull owing a good sensitivity to varas proteins: application to Crerebros spinal fluid [J]. Clin Chem Act a, 1982, 112: 93.

[10] Xu Shuyun, Bian Rulian, Chen Xiu. Pharmacological Experiment Methodology [M]. 2nd Edition, Beijing: People's Medical Publishing House, 1991. 922.

[11] Halliw ell B, Gu tt ridge JMC. Free radicals in biology and medicine[M ]. 2nd ed. New York: Oxford University Press, 1989. 543-550.

[12] Wang Xiaowen, Jiang Xiaoyan, Wu Liya Yiming, et al. The scavenging effect of the total glycosides of cistanche on free radicals and the protection of OH· induced DNA damage [J]. Chinese Pharmaceutical Journal, 2001, 36(1): 29-31.

[13] Wu Yuling, Xu Guangyuan. Experimental study of doxorubicin-induced acute cardiotoxicity in mice and the protective effect of Vit E [J]. Journal of Dalian Medical University, 1991, 13(1): 22-27.

From: Chinese Library Classification Number: Q949. 752. 7; R978. 1; R-332 Document identification code: A Article number: 1009-5551 (2003) 01-0028-03

Fund Project: The Natural Science Foundation of Xinjiang Uygur Autonomous Region Science and Technology Commission (96814)

About the author: Wu Liya Yiming (1964-), female (Uyghur), master, lecturer, research direction: cardiovascular pharmacology.