CN104940180A

CN104940180A - Novel application of benserazide hydrochloride

Info

Publication number: CN104940180A
Application number: CN201510407527.0A
Authority: CN
Inventors: 何书英; 余新超; 吴杰; 方婷欢; 季璇馨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2015-07-09
Filing date: 2015-07-09
Publication date: 2015-09-30

Abstract

The invention discloses the effect of benserazide hydrochloride in the aspect of preventing and treating atherosclerosis. Through animal experiments, the function of the benserazide hydrochloride for preventing and treating atherosclerosis is researched, the research accords with the development direction of medicine modernization in China, and the benserazide hydrochloride has an important social meaning and potential economic benefits in the basic research field and the application research field.

Description

A kind of new purposes of Ro-4-4602.

Technical field

The present invention relates to biomedicine field, be specifically related to the preventive and therapeutic effect of Ro-4-4602. for atheromatosis.

Background technology

Ro-4-4602. records in Chinese Pharmacopoeia version (ChP 2010) in 2010, European Pharmacopoeia 7.0 editions (EP7.0) and Japanese Pharmacopoeia 16 editions (JP16).Ro-4-4602. is white or off-white color crystalline powder, soluble in water, is slightly soluble in ethanol, is insoluble to acetone, its character extremely unstable, very responsive to the change of solvent PH, light, temperature and humidity.Ro-4-4602. is periphery decarboxylase inhibitor, is mainly used in treatment Parkinson's disease (essential tremor paralysis), after encephalitis or be associated with the symptomatic parkinson (non-drug induccd paralysis agitans syndrome) of cerebral arteriosclerosis.

In recent years, along with the raising of people's living standard, the life style of people and dietary habit there occurs huge change, a large amount of absorptions of high lipid food, the minimizing day by day of daily exercise, add being on the increase of aging populations, the sickness rate of cardiovascular and cerebrovascular disease is cumulative year after year trend, and in the death notation in many countries and regions, occupy the first.

Atherosclerosis (atherosclerosis, AS) be that the common disease of middle-aged and elderly people is become reconciled and sent out a disease, it is the main pathological basis of cardiovascular and cerebrovascular disease, it is a kind of disease being major lesions with cell proliferation and degeneration, and the adhesion and aggregation such as atherosclerotic generation and hyperlipidemia, smooth muscle cell proliferation and damage, macrophage and mononuclear cell have substantial connection.Atherosclerotic hazardness is very big, can cause patient that angina pectoris, myocardial infarction, sudden cardiac death, peripheral arterial disease etc. occur, atherosclerosis become the dead head in the whole world because of, serious threat is constituted to the life and health of patient.Just because of atherosclerotic serious harm, so in recent years, atherosclerosis receives the extensive concern of society and people, becomes a study hotspot for atherosclerotic study medication.Excavate a medicine with good atherosclerosis prevention effect to ensureing that the life and health of patient is significant.

Summary of the invention

The present invention adopts the Ro-4-4602. of variable concentrations to treat the ApoE-/-mice of high fat atherosclerosis modeling, found that low dosage Ro-4-4602. (25mg/kg/d) significantly can reduce blood vessel wall atheromatous plaque area and the blood vessel wall macrophage deposition of ApoE-/-Gao fat atherosclerosis modeling mice, and effect is better than middle dosage Ro-4-4602. (50mg/kg/d) and high dose Ro-4-4602. (100mg/kg/d).

Here is pharmacological moieties test and result:

One, laboratory animal grouping

Dosage group (ApoE-/-mice 10,50mg/kg/d), Ro-4-4602. high dose group (ApoE-/-mice 10,100mg/kg/d) in blank group (wild type C57 mice 10), model control group (ApoE-/-mice 10), Ro-4-4602. low dose group (ApoE-/-mice 10,25mg/kg/d), Ro-4-4602..

Two, Atherosclerosis Model and administration process is set up

All mices are all raised in SPF level Animal House, and take the feeding manner of free choice feeding and drinking-water.After adopting normal feedstuff adaptability to feed 1 week to above-mentioned five groups of mices, to blank group mouse feeding normal feedstuff, to model control group, the equal feeding high lipid food of three groups of mices of Ro-4-4602. administration, Ro-4-4602. low dose group mouse peritoneal injection Ro-4-4602. 25mg/kg/d is given while High fat diet, dosage group mouse peritoneal injection Ro-4-4602. 50mg/kg/d in Ro-4-4602., Ro-4-4602. high dose group mouse peritoneal injection Ro-4-4602. 100mg/kg/d, the normal saline of blank group and model control group mice then lumbar injection respective volume.Successive administration process, after 8 weeks, is put to death mice, is got mouse aorta specimen, carries out HE dyeing and the experiment of macrophage immunity groupization.

Three, pathological section HE staining examine

Get mouse aorta root 0.5cm to be placed in 10% formalin and to fix 24h, conventional dehydration, specimen is with paraffin embedding, serial section, and thick 4 μm of sheet, carries out HE dyeing and microscopy to section.

Under light microscopic, visible blank group mouse aorta wall is by inner membrance, and middle film and adventitia composition, demarcate clear, thickness is homogeneous.Inner membrance table, by simple squamous epithelium, is internal elastic membrane under inner membrance.Middle film forms primarily of elastica, and adventitia is loose connective tissue, and visible fat, as Fig. 1.Under model control group mouse aorta light microscopic, there is obvious atheromatous plaque in local inner membrance protuberance, and speckle is primarily of the foam cell composition engulfing lipid, and foam cell surface visible a small amount of collagen fiber, smooth muscle cell, as Fig. 2.Under Ro-4-4602. low dose group mouse aorta light microscopic, rarely seen a small amount of foam cell deposition, does not form obvious speckle, as Fig. 3.In Ro-4-4602. under dosage group and high dose group mouse aorta light microscopic, intimal thickening obviously alleviates, and speckle number obviously reduces, but effect is not as Ro-4-4602. low dose group, as Fig. 4, Fig. 5.

Four macrophage immunity groupizations detect

Get mouse aorta root 0.5cm to be placed in 10% formalin and to fix 24h, conventional dehydration, specimen is with paraffin embedding, serial section, and thick 4 μm of sheet, carries out macrophage immunity groupization to section and detect.

Under light microscopic, visible blank group mouse aorta inwall is without brown color plaque deposition, also has no macrophage, as Fig. 6.Model control group mouse aorta inwall has obvious plaque deposition, and has a large amount of macrophages in speckle, as Fig. 7.Ro-4-4602. low dose group mouse aorta inwall is without obvious plaque deposition, and rarely seen a small amount of macrophage adhesion, as Fig. 8.Dosage group and high dose group mouse aorta inwall in Ro-4-4602., visible obviously speckle, be rich in macrophage in speckle, comparatively model group reduces, but effect is not as Ro-4-4602. low dose group, as Fig. 9, Figure 10.

Accompanying drawing explanation

Fig. 1 is blank group mouse aorta root HE dyeing microscopic examination figure.

Fig. 2 is model control group mouse aorta root HE dyeing microscopic examination figure.

Fig. 3 is Ro-4-4602. low dose group mouse aorta root HE dyeing microscopic examination figure.

Fig. 4 is dosage group mouse aorta root HE dyeing microscopic examination figure in Ro-4-4602..

Fig. 5 is Ro-4-4602. high dose group mouse aorta root HE dyeing microscopic examination figure.

Fig. 6 is blank group mouse aorta root macrophage immunity group microscopy figure.

Fig. 7 is model control group mouse aorta root macrophage immunity group microscopy figure.

Fig. 8 is Ro-4-4602. low dose group mouse aorta root macrophage immunity group microscopy figure.

Fig. 9 is dosage group mouse aorta root macrophage immunity group microscopy figure in Ro-4-4602..

Figure 10 is Ro-4-4602. high dose group mouse aorta root macrophage immunity group microscopy figure.

Detailed description of the invention

Embodiment one

1) high lipid food formula

15% Adeps Sus domestica, 2% cholesterol, 1.5% whole milk powder, 1.5% yolk powder, 0.2% sodium cholate+79.8% normal diet.

2) HE dyeing

(1) paraffin section routine dewaxing: dimethylbenzene (I) 15min → dimethylbenzene (II) (should be completely transparent) 10min; (2) descending graded ethanol soaks aquation step by step: 100% ethanol (I) 2min → 100% ethanol (II) 2min → 95% ethanol 2min → 80% ethanol 2min → tap water for a moment; (3) dye: distilled water develop a film carve → haematoxylin liquid dye core 5min → tap water a moment → 1% acidic alcohol differentiation 30s (carry slotting several under) → running water a moment → weak ammonia liquor aqueous solution anti-indigo plant several seconds → running water 10min → set to 0 in the aqueous solution of .5% Yihong and redye 10min; (4) ascending gradient ethanol dehydration step by step: tap water (differentiation Yihong) → 95% ethanol (I) 2min → 95% ethanol (II) 2min → 100% in a moment ethanol (I) 2min → 100% ethanol (II) 2min; (5) transparent: dimethylbenzene (I) 5min → dimethylbenzene (II) 5min; (6) fixing, mounting: under coverslip resinene fix, mounting.

3) immunohistochemical staining

1., after paraffin section completes, be first put in 60 DEG C, baking oven on brandreth and dry sheet 1h, lower paraffin wax can be spot-check after terminating and whether melt.

2. then dimethylbenzene I 15min, dimethylbenzene II 15min, dehydrated alcohol 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, H2O 5min, PBS 5min successively.Attention: liquid level all wants the tissue in dipped section.

3. add enough citrate buffer solutions in beaker, first allow liquid boiling (require boiling too violent), then add section, after 20min, beaker stops heating, allows its natural cooling, and then PBS washes, 3*3min.

4. section dries, and then traverse is in wet box, drips 3%H2O2, cover lid incubated at room 10min.

5.PBS washes, 3*3min.Section dries, and then closes biopsy tissues with 3%BSA, wet box incubated at room 30min.

6. section deblocking liquid, then drips first antibody, and wet 4 DEG C, box spends the night.

7. within second day, take out wet box, and be put in indoor 30min, allow its rewarming.

8.PBS washes, 3*3min.PBS is got rid of in section, and then section drips second antibody and to wet box incubated at room 20min.

9.PBS washes, 3*3min.PBS is got rid of in section, and dropping the 3rd antibody of then cutting into slices wets box incubated at room 20min.

10.PBS washes, 3*3min.PBS is got rid of in section, and then section drips freshly prepared DAB nitrite ion, rinses out DAB liquid after 3-5min with water.Haematoxylin is redyed, and washes haematoxylin with water after 1-3min.Attention: concrete dyeing time is determined according to practical situation.

11. section dehydrate.Program is as follows: H2O 3min, 75% ethanol 3min, 85% ethanol 3min, 95% ethanol 3min, dehydrated alcohol 3min, dimethylbenzene I 15min, dimethylbenzene II 15min.

12. treat that dimethylbenzene volatilizes, neutral gum mounting.

Claims

1. Ro-4-4602. is used for the medicinal usage of atheromatosis control.