Results of research on the mechanism of action of CEDEMEX in drug addiction treatment in China

EFFECT OF CEDEMEX ON β- ENDORPHINE CONTENT

AND DOPAMINE IN THE BRAIN OF MORPHINE ADDICTED Mice

Ta Hai Nguyen*Duong Tranh*Sheep Tieu Cuong*Ly Toan Phuong* Hoang Nhan Bin*

Nguyen Phu Kieu** Pham Kim Man** Luong Viet Cao** Tran Dinh Binh**

* Guangxi Nanning Medical University – China

** Institute for Research and Treatment of Serious Diseases - Hanoi - Vietnam

***Hue University of Medicine - Vietnam

[Summary ]

Purpose: Observe the effects of Cedemex on β-endorphin and dopamine content in the brains of morphine-addicted rats

Methods: Using morphine injection under the skin for 7 days in increasing doses, building a mouse model of morphine addiction. Using radioimmunoassay to determine β-endorphin content in the hypothalamus of the brain and pituitary gland of mice. Using fluorescence method to determine the accumbens dopamine content of mice.

Result:

(1) Compared with the group that stopped using morphine, the group that used medium and high doses of Cedemex had the ability to quickly recover β-endorphin in the hypothalamus and pituitary areas (P < 0.05). In which, Cedemex doses were used. On average, the ability to recover quickly is 50%, 60%, using high doses of Cedemex has the ability to recover 55%, 36%.

(2) Compared with the group that stopped using morphine, the group that used medium and high doses of Cedemex could reduce the level of dopamine accumbens content of morphine-addicted rats (P<0.05) by 10%, 17%.

Conclusion: Changes in β-endorphin and dopamine in the body are associated with morphine dependence and withdrawal symptoms. Cedemex has the ability to stimulate the recovery of β-endorphin and dopa peptide.

Keywords: Cedemex morphine-dependent rats with spontaneous withdrawal (withdrawal) of β-endorphin dopamine

In recent years, the number of drug addicts in the world has been increasing, especially the increasing rate of pure drug use, making drug dependence increasingly serious and dangerous. Drugs have harmed mental and physical health. Cedemex is a remedy made from a combination of more than 10 herbal medicines such as Hoang Lien, Rhododendron, Diaphora, prepared in the form of a year old, widely used clinically in Vietnam and highly effective in drug treatment. Recent studies have shown that endogenous opioid peptides, especially β-endorphin and dopamine, are highly dependent on morphine. Research through the active ingredient of the drug Cedemex on β-EP content in the hypothalamus and pituitary gland and the effect of dopamine accumbens content on morphine-addicted rats has laid the foundation as well as one more step. Further research on the mechanism and effectiveness of Cedemex in treating morphine dependence.

Materials and methods

I. Animals and subgroups

Wistar male rats 72, SPF, weight 190-210g, provided by Guangxi Medical University Experimental Animal Center (SCXK experimental animal breeding license No. 2003-0003, Animal use license SYKG experiment No. 2003-2005). Animals are raised in the same environment. During the raising process, animals freely eat and drink and live in an environment with natural light and dark rules. After the animals adapted to being raised for 7 days, using random selection method, divided into a comparison group that did not use morphine (N), a group that stopped using morphine (SM), and a group that continued to use morphine (GM). , group using high dose of Cedemex (CH), group using medium dose of Cedemex (CM) and group using low dose of Cedemex (CL), each group of animals consists of 12 animals.

II. Experimental drugs and laboratory equipment

Morphine saline injection: Manufactured by Drug Factory No. 1 in Shen Duong, number 20040604. Cedemex capsules: provided by the Project Team and the Vietnam Institute for Research on Serious Diseases. Before clinical research, mix 80 Cedemex capsules into 30ml of warm water, then measure the volume to 40ml, i.e. 1g.ml-1 combined with the intra-abdominal injection solution. Dosage: High dose 8g.kg-1.d-1, medium dose 4g.kg-1.d-1, low dose 2g.kg-1.d-1. Reagents used to determine radioimmunity: β-endorphin, test box to determine radioimmunity 125I provided by the Biological Sciences Research Department, Department of Neurology, Military Medical University II . In addition, common reagents are all made in China. Laboratory tools to determine radioimmunity: GC-2016-γ radioimmunity meter (Trung Giai Branch - Sang Tan Joint Stock Company Limited). Machine to determine dopamine content: Fluorescence machine to determine the content of neurotransmitters (Waters Company - USA).

III. Modeling method

In addition to the group that did not use morphine, other groups relied on injecting morphine under the skin of mice in increasing doses, from day 1 to day 6, with different doses of 5, 10, 20, 30, 40 each time. , 50 mg.kg-1, twice a day (9:00, 17:00), on the 7th day, continue to maintain the dosage of 50mg.kg-1 injected with morphine under the skin of mice. Add up the dose of 410mg.kg-1, and weigh the mice every other day. After weighing the mice on the morning of the 8th day, the group of mice addicted to morphine continued to inject morphine under the skin, the group that stopped using morphine was compared for the same amount of time, with the same principle of injecting physiological saline, high and medium doses of Cedemex. and low based on the dosage of 8g.kg-1.d-1, 4g.kg-1.d-1, 2g.kg-1.d-1 injected into the mouse abdomen. Continuously inject the drug for 4 days and weigh the mice every other day. After 2.4 days of injection, the dead mice were treated, the brain was removed, the 3 necessary brain parts were separated (hypocerebrum, pituitary gland, dopamine accumbens) and placed in the -80°C refrigerator.

IV. Determination of radioimmunity

1. Sample processing: (1) Take the brain and pituitary gland in the -80°C refrigerator and weigh. (2) After weighing, place the brain and pituitary gland into the test tube, add 1N Hcl, formaldehyde (the above operations are done in a cold bath). Then transfer 1.5ml into a centrifuge tube, set at room temperature for 100min; (3) Centrifuge for 20 minutes under 4°C 4000rpm, take the clear solution above the 0.8ml test tube, then add 0.8ml 1N NaOH and acid, shake well. Leave the solution in the -20°C refrigerator waiting for testing.

2. Determination of radioimmunity [2,3]: β-EP radioimmunity drug box provided by the Biological Sciences Research Department, Department of Neurology, Military Medical University II, using an immunometer radioactivity GC-2016-γ.

V. Fluorescence testing method to determine DA content[4]

Remove the brain tissue from the -80°C refrigerator, wash it with cold physiological saline, dry it with paper, then weigh it; Add 0.4 mol.L-1 1ml perchlorate solution, shake well, centrifuge at 4°C/ 12000 rpm, take the solution on the test tube, use 0.4mol.L-1 1ml perchlorate solution, 4°C/ 12000 rpm centrifuge for 5 minutes, then filter the solution through a 0.22µm membrane filter.

Spectral conditions: symmetry column C18 (4.6 x 150 mm, 5µm); Test wave length 290nm, flow rate 1.0 ml.min-1, thermometer 30°C; 10µl sample. Combine sodium acetate solution 8.202g, lemon acid 21.014g, sodium 0.108g, EDTA -2 Na 0.056g, dibutylamine 0.129g, methanol 150ml, distilled water 1000ml, finally use triethylamine to prepare pH = 4. After Then use the equation (Y = 27328X- 11359, r= 0.9985, 1~ 6ng) to process the numbers.

BECAUSE. Number crunching

Use SPSS13.0 statistical software for processing. The number x ± s is used to represent and compare the average value of each group using pairs or testing groups.

Conclude

I. Short-term Cedemex treatment restores β-EP in the hypothalamus and pituitary in morphine-addicted rats

The group using medium and high doses of Cedemex had the ability to quickly recover β-endorphin in the lower brain and pituitary areas (P < 0.05). Using the drug on days 2 and 4 clearly shows that the higher the dose of Cedemex, the more clearly the ability to restore β-endorphin in the hypothalamus and pituitary areas. In the group of mice using an average dose of Cedemex, the subcerebral area increased from 16% to 83.5%, the pituitary gland increased from 31.8% to 78.3%; The group using high doses of Cedemex continued with the movement of time, but the amplitude of the increase decreased, the subcerebral area decreased from 70.2% to 52.1%, the pituitary gland decreased from 56.4% to 15%. .7 %. But the drug Cedemex restores β-EP still clearly showing dose dependence. The level of β-EP on day 4 was higher than on day 2 and close to normal levels, proving that maintaining drug use can cause β-EP to recover to normal. Compared with the group that continued to use morphine, using low, medium and high doses of Cedemex could all restore β-EP in the hypothalamus and pituitary areas. See table 1 for results

Table 1 Short-term Cedemex treatment restores β-EP in the hypothalamus and pituitary in morphine-addicted rats (x ± s)

Note: Comparison of group that stopped using morphine * P < 0.05, ** P < 0.01; Comparison of groups continuing to use morphine Δ P<0.05, ΔΔP< 0.01

II. Effects of short-term treatment of Cedemex on dopamine accumbens content in morphine-addicted rats

In particular, in the reduction of dopamine accumbens content, comparing the group using medium dose Cedemex with the group stopping using morphine, there was a clear difference (P<0.05), the group using high dose Cedemex after administering the drug. 2 days compared to the group that stopped using morphine, there was a clear difference (p<0.05), after 4 days the difference was not obvious. See table 2 for results.

Table 2 Effects of short-term treatment of Cedemex on dopamine accumbens content in morphine-addicted rats

Discuss

Morphine addiction and its addictive mechanism are still not completely clear. Currently, it is believed that it is related to the inhibition of endogenous Opioid peptides (such as β-EP), which are considered nerve EPs, most of which exist in the central and peripheral areas of the body, and are considered a source of naturally fun. There are also other studies demonstrating that, once morphine use is discontinued, along with the appearance of withdrawal symptoms in morphine-dependent animals, the opposition of EP function and DA levels increase significantly. Therefore, it is currently believed that reduced EP levels are one of the mechanisms and animal resistance depends on morphine group. EP types of the central nervous system conduct DA levels to develop adaptive regulation, causing morphine withdrawal symptoms. But research and observations on these aspects are being gradually researched.

Based on observations of predicted changes in the content of different neurotransmitters and different brain regions in the past, combined with factors such as the difficulty of taking specimens, we have observed Effects of Cedemex on FP changes in the brains of morphine-addicted rats and dopamine content, and research on the mechanism and effects of Cedemex in detoxifying rats addicted to morphine. The key points of this experiment mainly show: 1. Simultaneously taking samples from different brain regions to test different neurotransmitters, especially accumbens is very small but the amount used in the experiment There are many, so it requires high technology. 2. Using the method of gradually increasing the dose of intra-abdominal injection, under conditions of sparing drug use, the mice become acutely addicted to morphine, and build a mouse model of morphine addiction. This method is simple to do, saves time and money, and is also easier to observe and more stable than the 5-day model. 3. On the 2nd and 4th days after the withdrawal, conduct behavioral observation tests, but such experiments have not yet been reported.

Based on the experimental results in Table 1, we can conclude that Cedemex (especially at moderate doses) has a certain effect on hormones and the synthesis of EP-hypocerebral-glandular junction. of mice addicted to morphine, but whether Cedemex has the ability to regulate EP content in the body as well as the effect of natural detoxification treatment on mice addicted to morphine, specifically the mechanism still needs to be researched in depth. furthermore. Results in Table 2 show that the group using medium and high doses of Cedemex caused the amount of DA in the brain to return to normal in stopping the withdrawal of morphine-dependent rats, showing a tendency to change the dosage. At the same time, the long-term effectiveness and effects of the group using high dose cedemex are not equal to the group using medium dose cedemex. Similarly, whether the drug Cedemex has the effect of regulating dopamine levels in morphine-addicted rats or not, its specific mechanism also needs to wait for further research. Summarizing the above results, we can conclude that Cedemex drug has a certain therapeutic effect in detoxifying morphine-addicted rats, especially when using cedemex drug at an average dose. But the specific mechanism of action in the treatment of drug addiction of cedemex from now on still needs to be further researched from the overall aspects of behavior, morphological organ organization, and transmitters. nervous, molecular cellular level.

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