CN111803554A

CN111803554A - Novel application of Huatuo Zaizao pills

Info

Publication number: CN111803554A
Application number: CN202010817720.2A
Authority: CN
Inventors: 宁娜; 潘卫东; 黄暨生; 刘特; 彭丹婷; 王楠楠; 耿春贤; 张春波; 蒋文庆; 王康华
Original assignee: Guangzhou Baiyunshan Qixing Pharmaceutical Co ltd
Current assignee: Guangzhou Baiyunshan Qixing Pharmaceutical Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-10-23

Abstract

The invention relates to a new application of Huatuo Zaizao pills. In particular to application of Huatuo reconstruction pills in preparing a medicament with the effect of relieving atherosclerosis inflammatory reaction; application of HUATUOZAIZAO pill in preparing medicine with immunoregulation effect; application of HUATUOZAIZAO pill in preparing medicine for preventing and treating ischemic cerebral apoplexy is provided. The invention finds that the Huatuo Zaizao pills have better immunoregulation effect, particularly blood vessel immunoregulation effect, can effectively relieve inflammatory reaction in the process of atherosclerosis formation and delay the development of atherosclerosis in experimental research on the Huatuo Zaizao pills. Meanwhile, atherosclerosis is the main cause and important pathological base of ischemic stroke, so the aim of treating Huatuo Zaizao pills is to reduce the risk of occurrence and recurrence of ischemic stroke.

Description

Novel application of Huatuo Zaizao pills

Technical Field

The invention relates to a new application of a traditional Chinese medicine preparation, in particular to a new application of Huatuo Zaizao pills.

Background

Atherosclerosis (AS) is a progressive process in which intima of affected arteries successively undergoes lipid accumulation, fibroplasia and calcium precipitation, leading to thickening and hardening of blood vessel walls, mainly affects large and middle arteries, is characterized by fibrous plaque and/or atheroma under the intima of the vessels, and finally can cause ischemic and hypoxic changes and a series of complications of corresponding tissues and organs AS the lesion progresses. Atherosclerosis is a common pathological basis for vascular adverse events such as coronary artery disease, hypertension, cerebral infarction, and peripheral artery disease.

In recent years, atherosclerosis has attracted a great deal of attention from experts in the cardiovascular and cerebrovascular field. Both domestic and foreign data indicate that nearly 1/3 asymptomatic adult patients have carotid plaques, and a meta-analysis in foreign countries has shown that the overall prevalence of carotid stenosis > 50% is 4.2%. Atherosclerosis is an important cause of ischemic stroke, and emboli of cerebral embolism are mostly from carotid artery, and the treatment of the emboli can improve symptoms and reduce the risk of ischemic stroke.

The pathogenesis of atherosclerosis has not yet been fully elucidated. The traditional treatment method aiming at atherosclerosis mainly achieves the purpose of preventing and treating atherosclerosis by reducing blood fat and reducing accumulation of lipid on intima of affected arteries. However, the method is not radical treatment. With the further research on the mechanism of atherosclerosis, the theory of injury-response reaction and chronic inflammation, the generation, development and evolution process of atherosclerosis are relatively comprehensively and systematically described. There is increasing evidence that atherosclerosis is an inflammatory disease, with endothelial cells of the arterial wall, SMCs reacting with Reactive Oxygen Species (ROS) and oxidized low density lipoprotein (ox-LDL), and activation of a number of inflammatory factors constituting the most prominent feature of the pathological changes in atherosclerosis, whereby inflammation spans the entire process from accumulation of atherosclerosis from neutrophils in the blood to the vessel wall to rupture of the atherosclerotic plaque. It is understood from the theory of traditional Chinese medicine that turbid phlegm and blood stasis are main pathological products and pathogenic factors of arteriosclerosis, and the treatment needs to be performed by both phlegm and blood stasis. Huatuo Zaizao pills have the effects of promoting blood circulation, removing blood stasis, eliminating phlegm, dredging collaterals, promoting qi circulation and relieving pain, and are mainly used for treating apoplexy convalescence and sequelae caused by phlegm stasis and obstruction of collaterals clinically. In addition, research finds that Huatuo Zaizao pills have the effect of preventing and treating hyperlipidemia. However, no reports about the influence of Huatuo Zaizao pills on the inflammatory response in atherosclerosis exist so far.

Disclosure of Invention

Based on the above, a new application of Huatuozaizao pills is needed.

The specific technical scheme is as follows:

application of HUATUOZAIZAO pill in preparing medicine for relieving atherosclerosis and inflammatory reaction is provided.

In one embodiment, the amelioration is a decrease in the level of an inflammatory factor in the atherosclerotic inflammatory response upon administration of a therapeutically effective amount.

In one embodiment, the inflammatory factor comprises at least one of CXCL1/KC, TNF-a, CXCL2/MIP-2, IL-4, CCL5/RANTES, CXCL16, IL-10, VEGF, IL-6, and CXCL12/SDF-1 a.

The invention also provides application of Huatuo reforger pills in preparing medicines with immunoregulation effect.

In one embodiment, the immunomodulation is vascular immunomodulation.

In one embodiment, the immunomodulation is activation of helper Th1 cells and helper Th2 cells.

In one embodiment, the immunomodulation is activation of at least one of macrophages type M1 and macrophages type M2.

In one embodiment, the immunomodulation is modulation of the expression of RNAM6A methyltransferase, a macrophage of M1 type, thereby inhibiting activation of NF κ B, a macrophage of M1 type.

The invention also provides application of Huatuo reforger pills in preparing medicines with the efficacy of preventing and treating ischemic stroke.

In one embodiment, the prevention and treatment of ischemic stroke is performed by relieving an atherosclerotic inflammatory response.

Compared with the prior art, the invention has the following beneficial effects:

the invention finds that the Huatuo Zaizao pills have better immunoregulation effect, particularly blood vessel immunoregulation effect, can effectively relieve inflammatory reaction in the process of atherosclerosis formation and delay the development of arteriosclerosis. Meanwhile, atherosclerosis is the main cause and important pathological basis of cerebral apoplexy, particularly ischemic cerebral apoplexy, so the aim of treating Huatuo Zaizao pills is to reduce the risk of occurrence and recurrence of ischemic cerebral apoplexy.

Drawings

FIG. 1 shows HUATUOZAIZAO pill pair ApoE^-/-Effect of mouse body weight:

(A) preventing weight change in mice on 91 days of dosing;

(B) body weight change in mice on 35 days of treatment administration;

FIG. 2 is HUATUOZAOPelleting pair ApoE^-/-Effect of biochemical indices of blood of mice:

(A) preventing the effects of 56 days of administration on FFA, vLDL, ox-LDL, HDL-C levels and AI in the serum of mice;

(B) effects of prophylactic administration for 63 days, therapeutic administration for 7 days on FFA, vLDL, ox-LDL, HDL-C levels and AI in mouse serum;

(C) effects of prophylactic administration for 91 days, therapeutic administration for 35 days on HDL-C levels and AI in mouse serum;

FIG. 3 shows HUATUOZAIZAO pill pair ApoE^-/-Effect of mouse immune indices:

(A) effects of prophylactic administration for 63 days, therapeutic administration for 7 days on IgM in serum;

(B) the influence of 70 days of preventive administration and 14 days of therapeutic administration on the ratio of Th1/Th2, Treg/Th17 and M1/M2 of mice;

FIG. 4 shows HUATUOZAIZAO pill pair ApoE^-/-Effects of mouse cytokines:

(A) effects of the prophylactic administration for 84 days and the therapeutic administration for 28 days on mouse serum cytokines CXCL1/KC, IL-4 and CCL 5/RANTES;

(B) effects of the prophylactic administration for 84 days and the therapeutic administration for 28 days on mouse serum cytokines TNF-alpha, CXCL16, IL-10 and CXCL12/SDF-1 alpha;

(C) effects of the prophylactic administration for 84 days and the therapeutic administration for 28 days on mouse serum cytokines CXCL2/MIP-2, VEGF and IL-6;

FIG. 5 shows HUATUOZAIZAO pill pair ApoE^-/-Effect of mouse pathology:

(A-H) aortic oil red O staining results and immunohistochemical examination fluorescent staining results of all groups of mice;

in fig. 1-5, "Huatuo" in each group refers to Huatuo Zaizao pill; comparing with a normal B6 control group, wherein '1' indicates that P is less than 0.05, and '2' indicates that P is less than 0.01; and normal ApoE^-/-For the control group, "3" indicates P < 0.05 and "4" indicates P < 0.01; comparing with the model group, "+" indicates that P < 0.05, "+" indicates that P < 0.01; compared with Huatuo Zaizao pill treatment administration group, the "#" represents that P is less than 0.05, and the "#" represents that P is less than 0.01; ③ and XinCompared with the atorvastatin prophylactic administration group (positive control group I), ". tangle-solidup" indicates that P is less than 0.05, and "solidup" indicates that P is less than 0.01; comparison with the simvastatin treatment group (positive control group II), ". DELTA." indicates that P is less than 0.05, and ". DELTA." indicates that P is less than 0.01;

FIG. 6 shows HUATUOZAIZAO pill pair ApoE^-/-Effects of pathological manifestations in mice:

(A) h & E staining of mouse aortic arch, magnification 100 ×; (a) (b), (c) is a high magnification field of view of the part indicated by the arrow, magnification is 200 ×;

(B) MASSON staining of mouse aortic arch, magnification 100 ×; (a) (b), (c) is a high magnification field of view of the part indicated by the arrow, magnification is 200 ×;

(C) statistics of aortic arch plaque area and lipid infiltration area in mice (. p < 0.05);

FIG. 7 shows HUATUOZAIZAO pill pair ApoE^-/-Influence of mouse blood lipid index (. p < 0.05);

FIG. 8 is HUATUOZAIZAO pill pair

Flow cytometry measurements of activity and inflammatory factor release (. p < 0.01,. p < 0.05);

FIG. 9 shows HUATUOZAIZAO pill pair ApoE^-/-Mouse

Effect of methylation level of RNA M6A in whole cells:

(A) qPCR assay of RNA methyltransferase mRNA expression levels (. # p < 0.01, # p < 0.01);

(B) performing immunofluorescence staining on an aortic arch part of the mouse;

(C) dot blot identification of methylation levels of global RNA M6A (. # p < 0.01, # p < 0.01);

FIG. 10 is HUATUOZAIZAO pill pair

Effect of cellular NF κ B expression levels:

(A) western blot detection results of the expression level of NF kappa B and phosphorylation modified products (p-NF kappa B) thereof in cell nucleus (p < 0.05);

(B) performing immunofluorescence staining on an aortic arch part of the mouse;

(C) RNA co-immunoprecipitation-PCR detection results (. about.p < 0.01; # p < 0.01);

(D) huatuo Zaizao pill for treating rheumatic arthritis

Intracellular NF κ B RNA m6A modifies mechanisms that relieve inflammation and atherosclerosis.

Detailed Description

The new application of Huatuo Zaizao pills of the invention is further explained in detail with reference to the specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Huatuo Zaizao pill is prepared from rhizoma Ligustici Chuanxiong, semen Strychni Pulveratum, fructus evodiae, Borneolum, etc. Has effects of promoting blood circulation for removing blood stasis, eliminating phlegm, dredging collaterals, activating qi-flowing and relieving pain, and can be used for treating apoplexy convalescence and sequela with phlegm stasis and obstruction of collaterals, with symptoms of hemiplegia, spasm and numbness, facial distortion, and slurred speech. Experimental research shows that Huatuo Zaizao pill has effects of dilating blood vessel, regulating blood lipid and hemorheology, and protecting and repairing damaged brain cells.

The pharmacological actions of Huatuo Zaizao pills mainly include: can reduce the infarct volume of the model animal; reducing brain cell apoptosis; inhibiting the synthesis of excitatory amino acids of toxic substances; can regulate the content of nitric oxide and endothelin in the local lesion of brain tissue and blood, and protect brain cells from toxic damage; improving the neurological score of stroke rats; the functional morphology of brain cells and the integrity of organelles can be protected during ischemia so as to increase the number of synapses; can increase the synthesis of nerve growth factor family substances such as brain tissue nerve growth factor, fibroblast growth factor, brain-derived nerve nutrition factor and the like; promoting proliferation, differentiation and migration of neural stem cells during stroke; selectively block the calcium channel of brain cells and synapse membranes thereof, and obviously inhibit the increase of calcium ions in the brain cells after ischemia, thereby protecting the brain cells from being damaged by calcium overload; reducing the levels of total cholesterol, triglyceride, low density lipoprotein and malondialdehyde in the model animal, and further reducing the blood fat of the model animal.

At present, the influence of Huatuozhuang pills on the inflammatory reaction of atherosclerosis is not reported. Compared with the traditional method, the purpose of preventing and treating atherosclerosis is achieved by reducing blood fat, and the method for preventing and treating atherosclerosis by relieving the inflammatory reaction of atherosclerosis is an atherosclerosis preventing and treating method which is completely different in mechanism and is more 'permanent'.

Therefore, the embodiment of the invention provides application of Huatuo Zaizao pills in preparation of a medicine with the effect of relieving the inflammatory reaction of atherosclerosis.

Specifically, based on the theory of traditional Chinese medicine, the Huatuo Zaizao pill is subjected to a great deal of research on the pharmacodynamic mechanism by combining with the systematic knowledge of atherosclerosis. Firstly, the pharmacological action of immunological activity of Huatuo Zaizao pills for preventing and treating atherosclerosis is preliminarily explored, and the preventive and therapeutic administration of Huatuo Zaizao pills is proved to have the effect of relieving the inflammatory reaction of atherosclerosis by observing aspects such as vascular lesion, inflammatory reaction, immune cell composition and the like, so that the progression of atherosclerosis is delayed, and the action of Huatuo Zaizao pills is mainly related to weight control, lipid regulation, cellular immunity enhancement, vascular inflammatory cytokine expression inhibition and aortic plaque tissue fat accumulation reduction.

Secondly, the action mechanism of Huatuo Zaizao pills for relieving the inflammatory reaction of atherosclerosis is deeply researched, and the pathological mechanism, the phenotypic mechanism and the epigenetic mechanism all show that Huatuo Zaizao pills can be used for treating atherosclerosisRemarkably relieving the disease course progression of atherosclerosis. Huatuo Zaizao pills not only can effectively relieve the pathological manifestations of atherosclerotic mice and improve the blood lipid index of atherosclerotic mice, but also have the capability of epigenetic regulation, can eliminate m6A modification of specific region of NF kappa B mRNA 3' UTR by regulating the expression of RNA methylation transferase METTL14 and METTL3 in macrophages, thereby influencing the stability of NF kappa B mRNA, finally leading to inflammatory macrophages

To achieve the effect of relieving the inflammatory reaction of atherosclerosis.

In one particular embodiment, the amelioration is a reduction in the level of an inflammatory factor in the atherosclerotic inflammatory response upon administration of a therapeutically effective amount.

By "therapeutically effective amount" is meant the amount of the formulation, the minimum amount necessary to ameliorate, cure or treat one or more symptoms of the disease or disorder. The "therapeutically effective amount" will vary depending on the particular state, disease, disorder or condition being treated and its severity, as well as the age, weight, physical condition and responsiveness of the individual being treated. Thus, one or more of these parameters can be used to select and adjust the therapeutically effective amount of Huatuozhuang pills.

In one specific embodiment, the inflammatory factor comprises at least one of CXCL1/KC, TNF-a, CXCL2/MIP-2, IL-4, CCL5/RANTES, CXCL16, IL-10, VEGF, IL-6, and CXCL12/SDF-1 a.

In one particular embodiment, the atherosclerotic inflammatory response is an immune inflammatory response that exists throughout the development of atherosclerosis.

Further, in one specific embodiment, the atherosclerosis refers to the formation of more obvious lipid stripes, foam cell aggregation and large-area obvious lipid plaque formation at the aortic root, namely the atherosclerosis mainly in the atheromatous plaque stage.

In one particular embodiment, the atherosclerosis is atherosclerosis, which is indicative of dyslipidemia.

The embodiment of the invention also provides application of the Huatuo Zaizao pills in preparing a medicine with an immunoregulation effect.

Immunoregulation refers to the recognition and elimination of antigenic foreign bodies by the body, and the maintenance of physiological dynamic balance and relatively stable physiological functions. Usually through interactions between immune cells and immune molecules in the immune system, and with other systems such as the neuroendocrine system, where necessary, this process can be mediated by drugs to maintain the body at the most appropriate level in the most appropriate form for the immune response.

In one particular embodiment, the immunomodulation is vascular immunomodulation.

In one embodiment, the immunomodulation is activation of helper Th1 cells, helper Th2 cells.

In a specific embodiment, the immunomodulation is activation of at least one of macrophages type M1 and macrophages type M2.

In one specific embodiment, the immunomodulation is modulation of the expression of M1-type macrophage RNAM6A methyltransferase, thereby inhibiting the activation of M1-type macrophage nfkb.

The embodiment of the invention also provides application of the Huatuo Zaizao pills in preparing a medicine with the efficacy of preventing and treating ischemic stroke.

Cerebral apoplexy refers to cerebrovascular accident, and cerebral infarction, hypertension, cerebral hemorrhage, aneurysm, arteriosclerosis, arterial vascular malformation, subarachnoid hemorrhage and the like can be frequently seen in clinic, and the diseases belong to the category of cerebral apoplexy. Cerebral apoplexy can be mainly classified into ischemic stroke and hemorrhagic stroke. Ischemic stroke is a general term for brain tissue necrosis caused by stenosis or occlusion of blood supply arteries (carotid and vertebral) of the brain and insufficient blood supply to the brain.

In one specific embodiment, the prevention and treatment of cerebral arterial thrombosis is performed by relieving an atherosclerotic inflammatory reaction.

The following are specific experimental studies, and the starting materials used in the examples are all commercially available unless otherwise specified.

1. Research on regulating effect of Huatuozhuang pills on vascular immune function of atherosclerosis mice

1.1 animal grouping and drug intervention:

male B6/JNju-Apoe^em1Cd82/Nju mouse (ApoE)^-/-Mice), 280 mice, SPF grade, 4-6 weeks old, body weight 14-24 g; male C57BL/6JNju mice (B6 mice) 40, SPF grade, 4-6 weeks old, body weight 16-22 g; purchased from Nanjing university-Nanjing biomedical research institute, laboratory animal production license number: SCXK (threo) 2015-0001. Huatuo Zaizao Wan (HTZZW, batch No. 17195) is provided by Guangzhou Baiyunshanqixing pharmaceutical Co., Ltd.

280 ApoE only^-/-Mice, divided into 7 groups of 40 mice each with random weight balance, were normal ApoE^-/-Control group, model group, HUATUOZAIZAO pill preventing administration low dose group, HUATUOZAIZAO pill preventing administration high dose group, simvastatin preventing administration group (positive control group I), HUATUOZAIZAO pill treating administration group, and simvastatin treating administration group (positive control group II); 40 mice, B6, served as normal controls. Control group of Normal B6, Normal ApoE^-/-The control group was fed with normal feed, and the same volume (0.1mL) of physiological saline was injected intraperitoneally 1 time per week for 8 weeks; feeding high-fat feed containing 1% of cholesterol and 10% of lard for molding in the model group and the mice of each administration group, and continuously molding for 8-13 weeks; at the same time, 30. mu.g/mouse (0.1mL) of Lipopolysaccharide (LPS) was intraperitoneally injected 1 time per week for 8 consecutive weeks. Except for Huatuo reproduced pill treatment administration group and simvastatin treatment administration group (positive control group II), after 8 weeks of modeling (T57), the gavage administration is started, 1 time per day and 5 weeks of continuous administration are carried out, and the other groups of animals are modeled and simultaneously fed with the gavage administration, 1 time per day and 8-13 weeks of continuous administration are carried out.

Dose of Huatuo Zaizao pills is 3.12 and 6.24g/kg (equivalent to 5.304, 10.608g crude drug/kg, according to conversion of body weight-body surface area, about equivalent to 1, 2 times of the adult clinical daily dose of 8 g/times x 3 times/day x 1 day which is 24 g), dose of simvastatin group (positive control) is 0.0026g/kg (according to conversion of body weight-body surface area, about equivalent to 1 time of the adult clinical daily dose of 0.02 g/times x 1 times/day x 1 day which is 0.02 g). The specific grouping and dose setting for each group are detailed in table 1.

TABLE 1 summary of group and dosage design

1.2 results of the experiment

1.2.1 Huatuo Zaizao Wan Pao ApoE^-/-Effect of mouse body weight

The body weight of each group of mice was measured 2 times per week starting on day 1 of molding. The results are shown in FIG. 1:

model animal body weight and normal B6 control group, normal ApoE^-/-The control group ratio was increased to various degrees. Compared with model group, HUATUOZAIZAO pill has low dosage and high dosage for preventing animal body weight decrease in different degrees and is in dosage dependent relationship; the weight of animals treated by Huatuo Zaizao pill is reduced significantly. The Huatuozaizaowan pill is prompted to obviously reduce the weight of mice by both prevention and treatment administration.

1.2.2 Huatuo Zaizao Wan Pao ApoE^-/-Effect of Biochemical indices of blood in mice

And detecting the blood biochemical indexes of the mice in each group. The results are shown in FIG. 2:

on the 56 th day of molding, the TC and HDL-C, LDL-C contents, the Atherosclerosis Index (AI) and the LDL-C/HDL-C contents of the model group are obviously increased, and the TG and FFA contents are obviously reduced; on the 63 th day of molding, the TC, HDL-C, LDL-C, AI and LDL-C/HDL-C of the model group are obviously increased, and the contents of TG, FFA, ox-LDL and IgM are obviously reduced; on the 91 st day of molding, the TC and HDL-C, LDL-C, FFA contents and AI and LDL-C/HDL-C contents of the model group are obviously increased, and the ox-LDL content is obviously reduced, which indicates that the blood lipid level of the mice after molding is continuously increased and the IgM is obviously reduced.

Compared with model group, HUATUOZAIZAO pill has obviously reduced contents of FFA, vLDL and ox-LDL, increased content of HDL-C, LDL-C, and reduced AI at

days

56 and 63; on day 91 of dosing, HDL-C levels increased and AI decreased. Compared with the model group, on the 7 th day of Huatuo Zaizao pill treatment administration, the ox-LDL content is obviously reduced; on day 35 of administration, the TG reduction level was significantly reduced, HDL-C, LDL-C level was increased, and AI was reduced. The Huatuozheng pill is prompted to obviously reduce the arteriosclerosis index when being applied for prevention and treatment.

1.2.3 Huatuo Zaizao Wan Pao ApoE^-/-Effect of mouse immune index

And detecting the immunity index of each group of mice. The results are shown in FIG. 3:

immunoglobulin m (igm): model group animals, normal B6 control group and normal ApoE on day 63 of model building and administration^-/-Compared with a control group, the IgM content is obviously reduced. Compared with the model group, the IgM contents of the Huatuo Zaizao pill prevention administration group, the simvastatin prevention administration group, the Huatuo Zaizao pill treatment administration group and the simvastatin treatment administration group are obviously increased;

immune cell typing: model group animals, normal B6 control group and normal ApoE on day 70 of model building and administration^-/-The Treg/Th17 ratio and the M1/M2 ratio of the control group are obviously increased compared with the model group; compared with model group, HUATUOZAIZAO pill has different degrees of activating auxiliary Th1 and Th2 cell, and M1 and M2 type macrophage after 70 days of prophylactic administration. Prompting the Huatuo Zaizao pill to prevent administration, activate immunocytes and regulate immunologic function.

1.2.4 Huatuo Zaizao Wan Pao ApoE^-/-Effect of mouse cytokines

The content of the inflammation/chemotactic factor of each group of mice is detected by a cytokine chip technology. The results are shown in FIG. 4:

on the 84 th day of model building and administration, compared with a normal B6 control group and a normal ApoE-/-control group, the contents of serum cytokines CXCL1/KC, TNF-alpha, CXCL2/MIP-2, VEGF, CXCL16, IL-10 and CXCL12/SDF-1 alpha are obviously increased, which indicates that the inflammation level of the animals is increased after model building, and the continuous high-level cytokines promote the development of atherosclerosis.

Compared with model group, HUATUOZAIZAO pill has effects of reducing serum cytokine CXCL2/MIP-2, CXCL12/SDF-1 alpha, TNF-alpha, VEGF, CXCL16, IL-4, IL-10, CCL5/RANTES content after 84 days of preventive administration; 28 days after Huatuo Zaizao pill treatment, the contents of serum cytokines CXCL1/KC, TNF-alpha, CXCL2/MIP-2, IL-4, CCL5/RANTES and CXCL12/SDF-1 alpha are obviously reduced. The Huatuozhuang pill is suggested to produce the anti-atherosclerosis effect by reducing different types of inflammatory factors in serum to different degrees.

1.2.5 Huatuo Zaizao Wan Pao ApoE^-/-Effect of mouse pathological tissue

Pathological tissues of each group of mice were examined. The results are shown in FIG. 5, Table 2:

on the 91 th day of model building and administration, compared with a normal B6 control group and a normal ApoE-/-control group, the positive immunohistochemical reaction of the aortic plaque tissue oil red O staining, a macrophage surface marker (F4/80), a B lymphocyte surface marker (CD19) and a T cell surface marker (CD3) is obviously increased, which indicates that the fat accumulation and the immune cell aggregation of the aortic plaque tissue of the mouse are enhanced after model building. Compared with model group, HUATUOZAIZAO pill can reduce aortic plaque tissue fat accumulation after 91 days of prophylactic administration and 35 days of therapeutic administration. Suggesting that Huatuozhuang pills can produce the function of resisting atherosclerosis by reducing the fat accumulation of aortic plaque tissues.

TABLE 2 Huatuo Zaizao Wan Pao ApoE^-/-Effect of aortic oil red O staining in mice (n ═ 8)

Note: firstly, adopting oil red O staining section: "0 (negative), 1+ (weak positive); 2+ (mild positive), 3+ (moderate positive), 4+ (strong positive) ". ② and normal ApoE^-/-Comparison of control group, "3" means P<0.05, "4" means P<0.01; (iii) comparing with the model group, "+" indicates P<0.05, ". indicates P<0.01; comparison with simvastatin group administered for prevention (positive control group I), ". tangle-solidup" indicates P<0.05, ". tangle-solidup" denotes P<0.01。

2. Effect of Huatuo Zaizao pills in relieving atherosclerosis inflammatory reaction and regulation and control mechanism research thereof

2.1 animal grouping and drug intervention:

male ApoE^-/-C57/BABL mice, SPF grade, 6-8 weeks old, mass (30 + -5) g, total 40Only, purchase in shanghai's square model biology research center, license number: SCXK (Shanghai) 2017 and 0004. Huatuo Zaizao Wan (HTZZW, batch No. 17195) is provided by Guangzhou Baiyunshanqixing pharmaceutical Co., Ltd. ApoE^-/-C57/BABL mice, given general feed adaptive feeding for 1 week, were randomly divided into 4 groups: the animals were randomly divided into Blank Ctrl group (normal diet feeding), salt group (high fat diet feeding + equal volume Saline feeding), medium-dose HTZZW group (M) (high fat diet + HTZZW 8g/kg feeding), and low-dose HTZZW group (L) (high fat diet + HTZZW 4g/kg feeding), 8 each, and the intervention was performed for 12 weeks.

2.2 Experimental results:

2.2.1 Huatuo Zaizao Wan Pao ApoE^-/-Effect of pathological manifestations in mice

The pathological manifestations of the groups of mice are shown in figure 6:

h & E staining results show that the aortic root of a control group (Saline group) mouse has obvious lipid stripes, foam cells are gathered, large-area obvious lipid plaques are formed, even a certain number of plaques are endangered to rupture and are mainly in the atheromatous plaque stage. Compared with the Saline group, the AS plaque area of mice in the HTZZW dose group (M) group is obviously reduced, and the lipid deposition is obviously reduced.

MASSON staining results show that the proportion of collagen fibers at aortic root plaques of mice in Saline group is obviously increased. While HTZZW (M) the proportion of unstable collagen fibrils is significantly reduced at the aortic root plaques. Therefore, the Huatuo rebuild pills with medium dosage can obviously improve the vascular lipid deposition and the peripheral blood lipid level of AS mice.

2.2.2 Huatuo Zaizao Wan Pao ApoE^-/-Influence of mouse blood lipid index

The blood lipid index of each group of mice is shown in figure 7:

compared with the Blank Ctrl group, the peripheral blood serum of mice in the salt group has obviously increased levels of TG, TC and LDL-C, and obviously reduced levels of HDL-C. However, compared with Saline group, the Huatuo Zaizao pill has the effects that the mouse serum TG, TC and LDL-C values of the group are obviously fallen back and the HDL-C is also obviously increased. Experimental results show that Huatuo Zaizao pills can significantly improve the blood lipid index of AS mice fed by high-fat feed.

2.2.3 Huatuo Zaizao Wan Pao ApoE^-/-Mouse

Effect of Activity and inflammatory factors

Of each group of mice

The activity and inflammatory factors appeared as shown in figure 8:

FCM detection results show that in peripheral blood of mice in Blank Ctrl group,

and

the ratio of (a) was very low, whereas the ratio of the above two cells in the salt group was significantly increased; however, after treatment of the AS mouse model with a medium dose of HTZZW,

the proportion of cells is remarkably reduced

The cell proportion is significantly increased. The results suggest that HTZZW can significantly stimulate AS mice

To the direction of

And (4) transformation. Meanwhile, FCM detection results also show that the cell ratios of F4/80+/IL-1 beta + and F4/80+/IL-6+ are obviously reduced after the AS mouse model is treated by the HTZZW with medium dose. Research results suggest that Huatuozhuang pills can significantly reduce the activity of inflammatory macrophages (Macrophage) of AS mice and the release of inflammatory factors.

2.2.4 Huatuo Zaizao Wan PairApoE^-/-Mouse

Effect of cellular Whole RNA M6A methylation level

Each group of mice

The methylation level of RNA M6A in whole cells is shown in FIG. 9:

the expression level of the enzyme that manipulates the methylation of RNA M6A was measured for each set of samples. The qPCR results showed that mice in Saline group

In the cells, the expression level of the RNA M6A methyltransferase Mettle3 and Mettle14 is obviously increased, while the expression level of the RNA M6A demethylase Fto is obviously reduced. While the mice in the group treated with medium-dose HTZZW

In cells, the expression levels of Mettle3 and Mettle14 were significantly reduced. Meanwhile, the immunofluorescent staining results were also consistent with the above results.

The Dot blotting results show that the mice in the group were treated with a medium HTZZW dose

The overall M6A modification level of cellular RNA was significantly lower than that of the Saline group. Experimental results show that Huatuozhuang pills reduce AS mice by inhibiting the expression of RNA M6A methyltransferase

The methylation modification level of RNA M6A of the whole cells.

2.2.5 Huatuo Zaizao Wan Pao ApoE^-/-Effect of mouse NF-. kappa.B expression level

The NF κ B expression levels of the mice in each group are shown in fig. 10:

western blot results show that mice in Saline group

In the nucleus, the protein levels of total NF κ B and phosphorylated NF κ B (p-NF κ B) were significantly higher than the Blank Ctrl group. Of mice in the medium HTZZW-treated group

In the nucleus, protein levels of total and phosphorylated nfkb (p-nfkb) are significantly reduced. The IF staining results were also consistent with the Western blot results. Experimental results suggest that Huatuo Zaizao pill can inhibit

Cells are activated for NF κ B.

In conclusion, Huatuo Zaizao pills have good vascular immunoregulation effect and can relieve the inflammatory reaction of atherosclerosis, thereby achieving the curative effects of relieving the progression of arteriosclerosis and preventing ischemic stroke.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Application of HUATUOZAIZAO pill in preparing medicine for relieving atherosclerosis and inflammatory reaction is provided.

2. The use of claim 1, wherein the amelioration is a decrease in the level of inflammatory factors in the atherosclerotic inflammatory response upon administration of a therapeutically effective amount.

3. The use according to claim 2, wherein said inflammatory factor comprises at least one of CXCL1/KC, TNF-a, CXCL2/MIP-2, IL-4, CCL5/RANTES, CXCL16, IL-10, VEGF, IL-6 and CXCL12/SDF-1 a.

4. Application of HUATUOZAIZAO pill in preparing medicine with immunoregulation effect is provided.

5. The use of claim 4, wherein the immunomodulation is vascular immunomodulation.

6. The use of claim 4, wherein said immunomodulation is by activating at least one of helper Th1 cells and helper Th2 cells.

7. The use of claim 4, wherein said immunomodulation is activation of at least one of M1-type macrophages and M2-type macrophages.

8. The use of claim 4, wherein the immunomodulation is by modulation of the expression of M1-type macrophage RNAM6A methyltransferase, thereby inhibiting activation of M1-type macrophage NF κ B.

9. Application of HUATUOZAIZAO pill in preparing medicine for preventing and treating ischemic cerebral apoplexy is provided.

10. The use according to claim 9, characterized in that the prevention and treatment of ischemic stroke is by alleviating the inflammatory response of atherosclerosis.