Pharmaceutical composition for treating vascular dementia

Abstract

The invention provides a pharmaceutical composition for treating vascular dementia. The pharmaceutical composition is prepared from ligusticum wallichii, angelica sinensis, rhizoma pinellinae praeparata, pericarpium citri reticulatae, rhizoma acori graminei, polygala tenuifolia, tabasheer, astragalus membranaceus, poria cocos, rhizoma atractylodis macrocephalae, lumbricus, radix paeoniae alba, the root of kudzu vine, cassia twig and honey-fried licorice root. The traditional Chinese medicine is prepared from 10 g of ligusticum wallichii, 10 g of angelica sinensis, 10 g of rhizoma pinellinae praeparata, 12 g of pericarpium citri reticulatae, 10 g of rhizoma acori graminei, 6 g of polygala tenuifolia, 10 g of tabasheer, 10 g of astragalus membranaceus, 15 g of poria cocos, 10 g of rhizoma atractylodis macrocephalae, 20 g of lumbricus, 15 g of radix paeoniae alba, 15 g of

Description

Technical Field

[0001] The invention relates to the field of medical care and health, and in particular to a pharmaceutical composition for treating vascular dementia. Background Art

[0002] Vascular dementia (VD) is a severe cognitive dysfunction syndrome caused by low perfusion of brain areas such as memory, cognition and behavior due to various brain vascular factors. It is the second largest type of dementia after Alzheimer's disease. At the same time, VD is also the only type of dementia that can be prevented and treated so far, and early treatment is reversible. At present, the incidence of vascular dementia is gradually increasing. According to a report based on epidemiological data from the World Health Organization, about 55 million to 60 million people in the world suffer from vascular dementia, and it is expected to reach 85 million to 90 million by 2030. As the disease progresses, severe cases will completely lose the ability to live independently, bringing huge mental and economic burdens to families and society. How to effectively prevent and treat VD, alleviate the morbidity, and reverse the disease has become an important research topic in the industry at home and abroad. The pathogenesis of VD is complex. Modern medicine is mostly limited to a certain pathogenesis and has a relatively clear targeted medication. It does not significantly improve the overall function and daily living ability of VD patients, which undoubtedly limits the treatment of VD. Therefore, traditional Chinese medicine highlights its characteristics and advantages with its holistic concept and syndrome differentiation and treatment. In recent years, more and more evidence has shown that Chinese medicine may be a better choice for the treatment of VD. Summary of the invention

[0003] The purpose of the present invention is to provide a pharmaceutical composition for treating vascular dementia, aiming to improve the research on the pathological mechanism of VD and provide new ideas and methods for the treatment of the disease.

[0004] The present invention is achieved in this way: a pharmaceutical composition for treating vascular dementia, which is composed of chuanxiong, angelica, pinellia, tangerine peel, calamus, polygala, radix jasminoides, astragalus, tuckahoe, atractylodes, earthworm, white peony root, kudzu root, cassia twig and roasted licorice; the chuanxiong is 10g, angelica is 10g, pinellia is 10g, tangerine peel is 12g, calamus is 10g, polygala 6g, radix jasminoides 10g, astragalus is 10g, tuckahoe is 15g, atractylodes is 10g, earthworm 20g, white peony root is 15g, kudzu root is 15g, cassia twig is 10g and roasted licorice is 6g.

[0005] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the phlegm-resolving, blood-activating and orifice-inducing prescription can alleviate the learning and memory disorders caused by 2VO; the phlegm-resolving, blood-activating and orifice-inducing prescription can alleviate the pathological damage of neurons in the CA1 region of the hippocampus; the effect of the phlegm-resolving, blood-activating and orifice-inducing prescription on the expression of Akt and PI3K mRNA in the hippocampal tissue of VD rats; the effect of the phlegm-resolving, blood-activating and orifice-inducing prescription on the expression of p-Akt, PI3K and mTOR proteins in the brain tissue of VD rats. DETAILED DESCRIPTION

[0006] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0007] The VD rat model was established by the modified bilateral common carotid artery permanent ligation (2-VO) method in rats. The rats were randomly divided into sham operation group, model group, Chinese medicine group (Huatan Huoxue Kaiqiao prescription) and Western medicine group (oxiracetam), with 10 rats in each group. After 28 days of administration, the Morris water maze test was used to examine the memory ability, the hematoxylin-eosin (HE) staining method was used to observe the changes in the CA1 area of ​​the rat hippocampus tissue, and the real-time fluorescence quantitative PCR (RT-qPCR) method and protein immunoblotting (Western blot) method were used to detect the mRNA and protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in the hippocampus tissue of rats.

[0008] Forty rats were randomly divided into sham operation group, model group, oxiracetam group, and Huatan Huoxue Kaiqiao prescription group, with 10 rats in each group. The VD rat model was established by 2-VO method according to the literature method. This is a classic and useful CCH animal model. All rats were anesthetized with 3% sodium pentobarbital (0.2 ml / kg, IP). Without stimulating the vagus nerve, the bilateral common carotid arteries of four groups of adult male SD rats were gently separated from the carotid sheath with a glass rod, double ligated with 4-0 surgical sutures, and cut between the ligatures. The sham operation group rats also underwent bilateral common carotid artery operation after anesthesia, but the arteries were not ligated or blocked. The body temperature of SD rats was maintained at 37±0.5℃ during the operation. Gavage was started on the second day after surgery. The drug concentration and effective dose were converted according to the body surface area of ​​clinical humans and rats [doi:10.1007 / s12035-018-0906-2. Epub 2018 Mar1.], and the gavage dose for rats was 12.15 g·kg-1·d-1. The sham operation group and the model group were given normal saline, and the oxiracetam group was given 216 mg·kg-1·d -1 of oxiracetam solution. Each group was gavaged with an equal volume, 10 mL·kg-1 per day, once a day, for 28 consecutive days.

[0009] MT-200 Morris water maze (Shanghai Jiliang Software Technology Co., Ltd.); imaging system (Nikon DS-U3, Nikon, Japan); desktop high-speed refrigerated centrifuge (Xiangyi, H1850R); fully automatic gel imaging analysis system (Champ Gel 5000, Saizhi); LineGene 9600 Plus fluorescence quantitative PCR detection system (Bio-Ri, FQD-96A); micro-spectrophotometer (Merrill Lynch, SMA2000); small vertical electrophoresis instrument (BioRad, 1658001); membrane transfer instrument (BioRad, 170-4070); chemiluminescence imaging system (Minichemi 610, Saizhi); optical microscope (Nikon DS-U3, Nikon, Japan).

[0010] The Morris water maze is a black circular pool (160 cm in diameter and 45 cm in height) filled with water at 23 ± 1°C. The pool is equally divided into 4 parts, with an escape platform (10 cm × 10 cm) quadrant at a fixed center 2 cm below the water surface. First, the rats were trained 4 times a day for 5 consecutive days. In each training trial, the rats were gently placed in the water, facing the pool wall, in one of the four starting positions and allowed to swim freely to the escape platform. This procedure was repeated in a different order at all four starting positions each day. The escape latency (time to reach the underwater platform), swimming route to the underwater platform, and swimming speed of each trial were recorded by a computer video tracking system. If the rat reached the platform within 120 s, it was allowed to stay on the platform for 10 s for orientation. The rats that failed were gently guided to the platform by the experimenter and were allowed to stay there for 10 s. The rats were then removed and placed in a heated cage to rest until the next trial. On day 6, the memory retention test was performed without the platform. Each rat was placed in the quadrant opposite the target quadrant (the quadrant where the platform was located) and allowed to swim freely for 120 s. The time spent in each quadrant was recorded.

[0011] After behavioral assessment, rats were euthanized by deep anesthesia and exsanguination. Brain tissue was quickly placed on ice, and longitudinal sections of the brain were fixed with 4% paraformaldehyde. According to the manufacturer's instructions, brain samples between the optic chiasm and the mastoid body were removed, and 5 um serial coronal sections were prepared for HE staining. Two slides were selected from the same site of each rat and observed under an optical microscope (Nikon DS-U3, Japan).

[0012] After the water maze experiment, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and then quickly decapitated, placed on an ice table to quickly separate the hippocampus, and stored in liquid nitrogen at -80°C. Five rats were collected from each group. Total RNA in tissue samples was extracted with Trizol, and 5 μl of RNA was electrophoresed on 1% agarose gel to detect the integrity of RNA; reverse transcription was performed using Mona Bio's MonScriptTMRTIII All-in-One Mix with dsDNase kit, and the reverse transcription kit was operated according to the reaction conditions: 50°C for 25 min, 75°C for 5 min, the reaction was terminated, cooled on ice, and stored at -80°C for later use. PCR primers were designed and synthesized by Invitrogen, with 5'-ATCGTGTGGCAAGATGTGTATGAG-3' for Akt upstream and 5'-AGGCGGCGTGATGGTGATC-3' for downstream, and 126 bp in length, and 5'-TGCCTCCATTCACCACCTCTG-3' for PI3K upstream and 5'-CCTCTCCTTCCAAGCCTCAGTG-3' for downstream, and 80 bp in length. The PCR kit was used to operate, with 10 μL of MonAmp SYBR GreenqPCR Mix, 0.4 μL of PCR primer F and 0.4 μL of PCR primer R, 1 μL of cDNA template, and RNase Free Water to dilute the reaction system to 20 μL. The reaction procedure was 95°C pre-denaturation for 1 min, and three-step amplification was performed in a Real-time PCR instrument under the conditions of 95°C for 15 s, 60°C for 30 s, and 75°C for 30 s, for a total of 40 cycles. After the reaction was completed, a melting curve analysis was performed to exclude nonspecific amplification. The 2-△△CT method was used for relative quantitative analysis of the data.

[0013] Five rats were randomly selected from each group and anesthetized, and brain tissue was quickly isolated. The hippocampus was quickly isolated on ice and then quickly frozen at -80℃ for subsequent use. The hippocampus was homogenized with radioimmunoprecipitation lysis buffer. The proteins were then separated and transferred to PVDF membranes using 10% SDS-PAGE. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated with primary antibodies at 4℃ overnight. The antibodies were PI3K (1:500,), p-AkT (1:20000), mTOR (1:20000), and β-actin (1:25000). After several washes at room temperature, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3 000 dilution) for 1 hour. Finally, the proteins were detected using a chemiluminescence imaging system.

[0014] SPSS 27.0 was used for experimental treatment, and one-way analysis of variance was used for comparison between groups. For further pairwise comparison, LSD method was used when the variance was equal, and Dunnett T3 method was used when the variance was unequal. P < 0.05 was considered statistically significant.

[0015] Experimental Results 1. The effect of Huatan Huoxue Kaiqiao recipe on spatial learning and memory ability of rats with vascular dementia. (See Figure 1 ) 2. Effects of Huatan Huoxue Kaiqiao Decoction on the morphological structure of hippocampus in VD rats. (See Figure 2 ) 3. Effect of Huatan Huoxue Kaiqiao Recipe on the expression of Akt and PI3K mRNA in hippocampus of VD rats. (See Figure 3 ) 4. Effect of Huatan Huoxue Kaiqiao Recipe on the expression of p-Akt, PI3K and mTOR proteins in brain tissue of VD rats. (See Figure 4 ) 5. Electrophoresis of Akt, PI3K, and mTOR protein expression in hippocampus tissue of VD rats. (See Figure 5 ) The following conclusions can be drawn from the above experimental data: 1. The prescription of resolving phlegm, activating blood circulation and opening the orifices can alleviate the learning and memory impairment caused by 2VO Compared with the sham operation group, the average escape latency of rats in each VD group was significantly prolonged, while the number of crossing the platform area and the time spent in the platform quadrant were significantly reduced (P < 0.05 or P < 0.01); compared with the model group, the average escape latency of rats in the oxiracetam group and the Huatan Huoxue Kaiqiao prescription group was significantly shortened, while the number of crossing the platform area was significantly increased (P < 0.05). There was no statistically significant difference between the Huatan Huoxue Kaiqiao prescription group and the oxiracetam group (P > 0.05). See Table 1.

[0016] 2. The prescription for resolving phlegm, activating blood circulation and invigorating the orifices can alleviate the pathological damage of neurons in the CA1 region of the hippocampus The hippocampus is closely related to learning and memory functions, and is one of the brain regions that is highly sensitive to ischemia and hypoxia. HE staining was used to observe the effect of Huatan Huoxue Kaiqiao Fang on hippocampal neuronal damage in VD rats. At 4 weeks after 2VO, the pyramidal neurons in the CA1 region of the hippocampus in the sham operation group were densely arranged, the neurons were clear, of moderate size, and had normal microstructures. The model group showed obvious pathological changes, with loosely arranged neurons, neuronal atrophy and loss, and light staining. The neuronal structure of the hippocampus in the oxiracetam group and Huatan Huoxue Kaiqiao Fang group was relatively complete, with a more regular morphology, and the pathological changes in the hippocampal tissue were significantly improved compared with the model group. See Figure 1 .

[0017] 3. Effect of Huatan Huoxue Kaiqiao Decoction on the mRNA expression of Akt and PI3K in hippocampus of VD rats Compared with the sham operation group, the expression of Akt and PI3K mRNA in the model group was significantly decreased (P<0.05); compared with the model group, the expression of Akt and PI3K mRNA in the oxiracetam group and the Huatan Huoxue Kaiqiao prescription group was significantly increased (P<0.05). See Table 2.

[0018] 4. Effect of Huatan Huoxue Kaiqiao Decoction on the expression of p-Akt, PI3K and mTOR proteins in brain tissue of VD rats Compared with the sham operation group, the expression of p-Akt, PI3K, and mTOR proteins in the hippocampus of rats in the model group was significantly decreased (P<0.05); compared with the model group, the expression of p-Akt, PI3K, and mTOR proteins in the oxiracetam group and the Huatan Huoxue Kaiqiao prescription group was significantly increased (P<0.05). Figure 2 , Table 3.

[0019] In summary, Huatan Huoxue Kaiqiao prescription has a neuroprotective effect on rats with cognitive dysfunction after experimental cerebral hypoperfusion, and its mechanism is to activate PI3K / Akt / mTOR signaling and inhibit neuronal death in the hippocampus.

[0020] The raw materials used in the experiment are as follows: 1. Animals Male Sprague-Dawley rats were obtained from Beijing Sibeifu Biotechnology Co., Ltd. (animal license number: SCXK (Beijing) 2019-0010) and maintained in a controlled environment with a standard temperature of 25 ± 2 °C and a humidity of 60 ± 10% with a 12 h light-dark cycle. Rats were given unlimited water and a standard rodent diet throughout the experiment. This project was reviewed and approved by the Medical Ethics Committee of Ningxia Hospital of Traditional Chinese Medicine.

[0021] 2. Medication All Chinese medicinal materials in the prescription were provided by the Chinese medicine pharmacy of Ningxia Hospital of Traditional Chinese Medicine. Distilled water was added to make a 100% suspension and stored at 4°C for later use. Oxiracetam capsules (0.4 g per capsule) were purchased from CSPC Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd. (National Medicine Approval No. H20031033).

[0022] 3. Reagents and Instruments Xylene (Shanghai Jiuyi Chemical Reagent Co., Ltd., batch number 1330-20-7); HE staining solution set (Servicebio, batch number G1003); neutral gum (Sinopharm Chemical Reagent Co., Ltd., batch number 10004160); TRIzon Reagent (Kangwei Century, batch number CW0580); MonScript RTIII All-in-One Mix with dsDNase (Monad, batch number MR05101); MonAmp SYBR Green qPCR Mix (Monad, batch number MQ10101); high-efficiency RIPA tissue / cell lysis buffer (Solabo, batch number R0010); BCA Protein Assay kit (Kangwei Century, batch number CW0014); SDS-PAGE gel preparation kit (Kangwei Century, batch number CW0022); PVDF membrane (GE, batch number 10600021); Ultrasensitive plus ECL chemiluminescent substrate (Zhongshi Tongchuang, batch number ZS-PRO-2005); protease inhibitor cocktail (Abmole, batch number M5293); phosphorylated protease inhibitor cocktail (Abmole, batch number M7528). It should be clear that the test methods used in the above examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used in the above examples are all commercially available unless otherwise specified.

[0023] Figure 1 To show the effect of Huatan Huoxue Kaiqiao prescription on spatial learning and memory ability of rats with vascular dementia. Figure 2 To show the effect of Huatan Huoxue Kaiqiao prescription on the morphological structure of hippocampus in VD rats. Figure 3 To show the effect of Huatan Huoxue Kaiqiao recipe on the expression of Akt and PI3K mRNA in hippocampus tissue of VD rats. Figure 4 To show the effect of Huatan Huoxue Kaiqiao prescription on the expression of p-Akt, PI3K and mTOR proteins in brain tissue of VD rats. Figure 5 Electrophoresis showing the expression of Akt, PI3K, and mTOR proteins in the hippocampus of VD rats.

Claims

1. A pharmaceutical composition for treating vascular dementia, the pharmaceutical composition is composed of Chuanxiong, Angelica sinensis, Pinellia ternata, Tangerine peel, Acorus calamus, Polygala tenuifolia, Radix dasyphyllae, Radix Astragali, Poria, Atractylodes macrocephala, Pheretima macrorrhizae, White Peony Root, Pueraria lobata, Cassia twig, and Radix Glycyrrhizae.

2. A pharmaceutical composition for treating vascular dementia according to claim 1, characterized in that: The described ingredients include 10g of Chuanxiong, 10g of Angelica sinensis, 10g of Pinellia ternata, 12g of Citrus reticulata, 10g of Acorus calamus, 6g of Polygala tenuifolia, 10g of Radix Glehniae, 10g of Radix Astragali, 15g of Poria, 10g of Atractylodes macrocephala, 20g of Pheretima, 15g of White Peony Root, 15g of Puerariae Radix, 10g of Cinnamon Twig, and 6g of Radix Glycyrrhizae Preparata.

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