CN115990187B - Traditional Chinese medicine extract for improving Alzheimer disease and application thereof - Google Patents
Traditional Chinese medicine extract for improving Alzheimer disease and application thereof Download PDFInfo
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- CN115990187B CN115990187B CN202211565719.0A CN202211565719A CN115990187B CN 115990187 B CN115990187 B CN 115990187B CN 202211565719 A CN202211565719 A CN 202211565719A CN 115990187 B CN115990187 B CN 115990187B
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Abstract
The invention belongs to the technical field of biological medicines, and particularly provides a traditional Chinese medicine extract for improving Alzheimer's Disease (AD), which comprises honeysuckle polysaccharide and forsythoside A. The traditional Chinese medicine extract provided by the invention consists of plant natural products, mediates cell functions through a nerve inflammation pathway/intestinal microecology dual-pathway, intervenes AD with the aim of reducing nerve inflammation, and can relieve Abeta 25‑35 The induced HT-22 cell injury promotes the formation and consolidation of mouse spatial memory, remarkably relieves AD pathological characterization, relieves the neuroinflammation in AD pathology by regulating and controlling the expression of IL-17 signal channel related genes, can also improve the cognitive dysfunction of an AD model mouse by regulating the abundance of specific species and promoting the increase of beneficial bacteria and correcting the disturbance of intestinal flora, has remarkable effect on improving AD, and is a safe and effective multi-target drug.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a traditional Chinese medicine extract for improving Alzheimer's disease and application thereof.
Background
Alzheimer's Disease (AD) is a common neurodegenerative disease, is one of the main reasons for senile dementia, and pathological features include senile plaques formed by deposition of extracellular beta amyloid (Abeta) in brain and neurofibrillary tangles formed by hyperphosphorylation of intracellular Tau protein, which are influenced by factors such as inheritance, environment and the like, and the AD has the characteristics of long latency, long disease course and the like, once the AD occurs, the AD is irreversible, and various treatment means can only delay the disease course development and cannot radically treat the AD. Currently, cholinesterase inhibitors and glutamate receptor antagonists (such as donepezil and memantine), targeted Abeta monoclonal antibodies, nonsteroidal anti-inflammatory drugs and the like are mainly used for treating AD, and the treatment has a certain effect, but the side effect is obvious, and the treatment is limited to certain symptoms, so that the optimal prevention and cure effects cannot be achieved. In order to overcome the defect of chemical medicine in treatment, plant natural products with the characteristics of multiple targets, less side effects and the like are widely concerned, and the development of AD-resistant plant medicines has become a current hot development direction.
Honeysuckle is a dry flower bud of honeysuckle (Lonicera japonica thunder.) belonging to the family Caprifoliaceae, has a long administration history in China, and has the effects of clearing heat and detoxicating, resisting virus and bacteria, resisting inflammation, enhancing immunity, scavenging free radicals, resisting endotoxin and the like. Fructus forsythiae (Forsythia suspensa) is a plant of the genus Forsythia of the family Oleaceae, and has antiviral, antibacterial, antioxidant and antiinflammatory effects. Honeysuckle and weeping forsythiae capsule are common drug pairs, and can enhance the efficacy of clearing heat and detoxicating and dispelling wind and heat by combining. In addition to the representative prescription of Yinqiao powder, more than 400 Chinese patent medicines related to honeysuckle-fructus forsythiae medicine pairs exist in the existing Chinese patent medicines on the market. Network pharmacological researches show that the honeysuckle and weeping forsythia 1:1 compatibility has stronger anti-inflammatory effect than single use; the material basis of the single decoction, the combination and the mixed decoction of the honeysuckle and the weeping forsythia in the medicine is proved to be changed; in addition, the honeysuckle and weeping forsythia medicine has promotion effect on dissolving polysaccharide.
The role of phenylethanoid glycosides and plant polysaccharides in the treatment of AD is now gradually demonstrated, whereas molecular interactions between polysaccharides and phenolic substances can affect bioavailability and beneficial effects. Forsythoside A is the main component of fructus forsythiae, belongs to phenethyl alcohol glycoside compounds, and has the functions of protecting nerves, improving memory injury and repairing neurons; the honeysuckle polysaccharide can improve the intestinal immune function of an immunosuppressive mouse by regulating intestinal flora and short-chain fatty acid generation. Therefore, combining the action and the mechanism which can be exerted in AD treatment, mediating the cell function through the nerve inflammation pathway/intestinal microecological dual-pathway, and taking the aim of reducing the nerve inflammation as the target, the intervention of AD is a research direction with great potential. The invention provides a traditional Chinese medicine extract, which mediates cell functions through a nerve inflammation pathway/intestinal microecology dual pathway and is used for intervening AD with the aim of reducing nerve inflammation.
Disclosure of Invention
The invention aims to provide a safe and effective multi-target drug for improving AD, so as to solve the problems of poor curative effect and larger side effect of the prior art that most of AD clinical therapeutic drugs are single-target chemical drugs.
For this reason, the invention provides a traditional Chinese medicine extract for improving Alzheimer's disease, which comprises honeysuckle polysaccharide, wherein the honeysuckle polysaccharide is prepared by the following method: pulverizing flos Lonicerae, extracting with ultrasonic water extraction method, centrifuging to obtain supernatant, concentrating, adding ethanol, standing, centrifuging to obtain precipitate, dissolving the precipitate with water, extracting to remove protein to obtain crude polysaccharide of flos Lonicerae; purifying the honeysuckle crude polysaccharide by column chromatography to obtain the honeysuckle polysaccharide.
Specifically, the extraction conditions of the ultrasonic water extraction method are that the ultrasonic time is 25-35min, the ultrasonic temperature is 55-65 ℃, and the ratio of feed to liquid is 1:25-1:35 in terms of volume-mass ratio.
Specifically, a Savag reagent is added in the preparation process of the honeysuckle polysaccharide for extraction and deproteinization; the Savag reagent comprises chloroform and n-butanol; the volume ratio of the chloroform to the n-butanol is 4:1.
specifically, in the preparation process of the honeysuckle polysaccharide, the honeysuckle crude polysaccharide is purified by a DEAE-52 cellulose chromatographic column and a Sephadex G-100 gel chromatographic column to obtain the honeysuckle polysaccharide.
Specifically, the traditional Chinese medicine extract also comprises forsythoside A; the mass concentration ratio of the forsythoside A to the honeysuckle polysaccharide is 1:2 to 1:6.
Specifically, the preparation method of the forsythoside A comprises the following steps: pulverizing folium forsythiae, placing in a container, leaching with ethanol solution, adsorbing with resin, eluting, and treating the eluate with reversed phase chromatography to obtain forsythoside A.
Specifically, the concentration of ethanol in the extraction process is 40-50%, the liquid-material ratio is 25:1-30:1 by volume-mass ratio, the extraction temperature is 40-60 ℃, and the extraction time is 20-30min.
Specifically, in the adsorption and elution processes, AB-8 macroporous adsorption resin is used as an adsorbent, 5-10BV of water is used for eluting, then 5-10BV of 30% ethanol solution and 5-10BV of 50% ethanol solution are used for eluting respectively, and the eluting flow rate is 2-5BV/h.
The traditional Chinese medicine extract provided by the invention can be used for preparing medicines for improving AD.
Specifically, the medicine for improving AD takes the mixture of honeysuckle polysaccharide and forsythoside A or the honeysuckle polysaccharide as an active ingredient, and also comprises pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the traditional Chinese medicine extract provided by the invention mediates cell functions through a nerve inflammation pathway/intestinal microecology dual pathway, and intervenes AD with the aim of relieving nerve inflammation, thereby relieving Abeta 25-35 The induced HT-22 cell injury promotes the formation and consolidation of mouse spatial memory, remarkably relieves AD pathological characterization, relieves the neuroinflammation in AD pathology by regulating and controlling the expression of IL-17 signal channel related genes, can also improve the cognitive dysfunction of an AD model mouse by regulating the abundance of specific species and promoting the increase of beneficial bacteria, and corrects intestinal flora disorder, thereby having remarkable effect on improving AD. In addition, the traditional Chinese medicine extract is composed of natural plant products, is a safe and effective multi-target medicine, and solves the problems of poor curative effect and large side effect of single-target chemical medicines.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 shows the cytotoxicity of FA and/or LP and their binding to Abeta in example 2 of the present invention 25-35 Effect of induced HT-22 cell damage; a. b and c: statistical analysis of cell viability of FA and/or LP on HT-22 cells; d. e and f: FA and/or LP vs. Abeta 25-35 Statistical analysis of cell viability of induced HT-22 cell death.
FIG. 2 shows the results of forsythoside A, honeysuckle polysaccharide and mixtures thereof vs. Abeta in example 2 of the present invention 25-35 Results of the effects of induced HT-22 cell mitochondrial membrane potential.
FIG. 3 is the experimental results of the spatial exploration of the mice in example 3 of the present invention; a, experimental design; b, recording infrared tracks; c, positioning the time required by the navigation experiment mouse to find the platform; d: the number of times the mouse passes through the quadrant where the platform is located in the space exploration experiment; e, exploring the swimming path of the mouse searching platform in the experiment in space; f: space exploration of the time that mice remained in the target quadrant in the experiment.
FIG. 4 shows the effect of forsythoside A, honeysuckle polysaccharide and mixtures thereof on Abeta-induced HT-22 cell and the inflammatory factor content in mouse serum in example 3 of the present invention.
FIG. 5 shows the effects of forsythoside A, honeysuckle polysaccharide and mixtures thereof on Abeta in the hippocampus and cortex of the brain of a mouse in example 3 of the present invention 25-35 Protein content effects of immunofluorescence reactions.
FIG. 6 is a KEGG biological process class of mouse brain tissue differentiation genes of MIX group and APP/PS1 group in example 4 of the present invention.
FIG. 7 is a graph showing the verification of differential gene expression levels in brain tissue of mice in example 4 of the present invention; PCR verification of the expression levels of Il-17a, act1, ifkba, nfkbp65 and cox 2; b is Western blot verification of NF kappa Bp65 and IL-17A; c is a Western blot quantification chart.
FIG. 8 shows the effect of honeysuckle polysaccharide in combination with forsythiaside A on the alpha diversity and structure of intestinal flora of AD mice in example 5 of the present invention.
FIG. 9 shows dominant species distribution of intestinal microbiota of AD mice in example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in the following examples, and it is obvious that the described examples are only some examples of the present invention, but not all examples. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the invention pertains will appreciate that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The effects of the pharmaceutical composition of the present invention and its use are examined below by way of specific examples.
Example 1:
the embodiment provides a traditional Chinese medicine extract for improving Alzheimer disease, which comprises honeysuckle polysaccharide and forsythoside A, wherein each component is obtained through the following steps.
The weeping forsythia leaves used in the embodiment are obtained from An Zexian of Shanxi province, the honeysuckle flower samples are obtained from giant deer county of Hebei province, and the weeping forsythia leaves and the honeysuckle flowers are respectively identified as weeping forsythia leaves of the forsythia genus of Oleaceae family, are dried and then stored at a low temperature.
(1) Extraction and purification of honeysuckle polysaccharide
Pulverizing dried flos Lonicerae, sieving with No. 4 sieve, extracting with ultrasonic water extraction method for 30min at 60deg.C, and mixing the materials at a volume/mass ratio of 1:30. Centrifuging the filtrate to obtain supernatant, concentrating to a proper volume, adding 95% ethanol to make ethanol content 80%, standing for 48 hr, centrifuging to obtain precipitate. Dissolving the precipitate with water, adding Savag reagent (chloroform: n-butanol=4:1), extracting to remove protein, and repeating the operation until no denatured protein is produced. Concentrating deproteinized polysaccharide solution to a proper volume, and freeze-drying to obtain crude honeysuckle polysaccharide. Dissolving the crude polysaccharide sample of the honeysuckle in water, fully dissolving, loading the solution on a DEAE-52 cellulose chromatographic column and a Sephadex G-100 gel chromatographic column, and purifying the crude polysaccharide to obtain purified honeysuckle. The yield of the purified honeysuckle polysaccharide is 1.15%, the polysaccharide content in the honeysuckle polysaccharide is 96.31%, and the monosaccharide composition of the honeysuckle polysaccharide obtained by gas chromatography and mass spectrometry identification is arabinose, rhamnose, ribose, xylose, mannose, fructose, galactose and glucose.
(2) Extraction and purification of forsythoside A
Pulverizing fructus forsythiae leaf, placing in a container, leaching with 46% ethanol solution, and mixing the materials at a liquid-material ratio of 29:1 (mL: g), extraction temperature was 51℃and extraction time was 28min. Then, the AB-8 macroporous adsorption resin is used as an adsorbent for adsorption, 8BV of water is used for elution, and then 6BV of 30% ethanol solution and 6BV of 50% ethanol solution are used for elution respectively, wherein the elution flow rate is 3BV/h. And finally, treating the eluent by adopting a C18-silica gel column reversed phase chromatography to obtain the purified forsythoside A. The extraction rate of forsythoside A is 5.95%, and the purity is 96%.
Example 2:
Aβ 25-35 is one of main types of neurotoxicity caused by Abeta, in vivo experiments show that the Abeta can lead to memory decline and the occurrence of brain inflammation, and research shows that the Abeta 25－35 The induced pro-inflammatory cytokines such as IL-1 beta and TNF-alpha can cause neuronal apoptosis, and have close relation with the pathogenesis of AD. This example investigated the pair A beta of honeysuckle polysaccharide and forsythoside A provided in example 1 25-35 Improvement of induced HT-22 nerve cell injury.
1. Cell culture
HT22 mouse hippocampal neuronal cells (337709;BeNa Culture Collection,China) were cultured in medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1%100U/mL penicillin and 100. Mu.g/mL streptomycin at 37℃and 5%/95% CO 2 Air.
2. Cytotoxicity of cells
HT22 cells at 4.0X10 4 Cell/well density was seeded into 96-well plates, pretreated with Forsythoside A (FA) and/or honeysuckle polysaccharide (LP) at varying concentration ratios for 3h, then with Abeta 25-35 (Sigma-Aldrich, USA) for 24h. Subsequently, 10. Mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) (5 mg/ml) was added to each well and incubated for 4h in the dark. Finally, 100 μl DMSO was added to each well to dissolve the fuma crystals. To analyze cell viability, synergy was used TM 4Microplate Reader (BioTek Instruments, USA) the optical density of each well was analysed at 490 nm.
As shown in FIG. 1, the safe concentration of forsythoside A is 20-100 μg/mL, and the safe concentration of honeysuckle polysaccharide is 100-500 μg/mL. As shown in FIG. 1, in the safe concentration range, both forsythoside A and honeysuckle polysaccharide are treated independently to improve Abeta 25-35 The induced HT-22 cell injury, the combination of forsythoside A and the honeysuckle polysaccharide is not obvious toxic to cells, and the Abeta is improved 25-35 Induced HT-22 cell damage.
3. Mixing ratio of forsythoside A and flos Lonicerae polysaccharide
Forsythoside A and gold and silverThe flower polysaccharide is mixed in a cell experiment system according to various ratios (1:1 to 1:10) to screen the optimal concentration ratio, and the result is shown in table 1, when the mass concentration ratio of forsythoside A to honeysuckle polysaccharide is 1:5, EC of 50 The value is minimum, which indicates that the action effect is strongest.
TABLE 1 FA/LP mixture vs. Abeta 25-35 Inhibition of induced HT-22 cell damage (in EC 50 Representation of
Data are mean ± standard deviation, a, b, c, d significant differences for each group comparison are indicated by different letters, P <0.05.
4. Mitochondrial Membrane Potential (MMP) detection
In order to verify the synergistic effect of forsythoside a and honeysuckle polysaccharide, both 1:5 ratio of mixture to Abeta 25-35 Effect of induced mitochondrial membrane potential effects of HT-22 cells.
HT22 cells at 4.0X10 4 Cell/pore density inoculating into 6-well plate, pretreating with forsythoside A and/or flos Lonicerae polysaccharide at different concentrations for 3 hr, and treating with Abeta 25-35 Incubate for 12h. Then, 2. Mu.M of 5,5', 6' -tetrachloro-1, 1', 3' -tetraethyl-imida-carbocyanine iodination staining solution (jc-1,420,200;EMD Millipore,USA) was irradiated in the dark for 20 minutes. Fluorescence microscopy and CCD camera (200 Xmagnification; TE2000; nikon, japan) were used and quantification was performed using imageJ software version 1.46. The ratio of the red-green fluorescence intensities was used as an indicator of MMP changes. As shown in figure 2, the forsythoside A and the honeysuckle polysaccharide mixture can significantly improve Abeta 25-35 The resulting decrease in cell membrane potential indicates that forsythoside A and honeysuckle polysaccharide are in Abeta 25-35 Synergistic remission may be exerted in induced HT-22 cell injury.
In conclusion, the honeysuckle polysaccharide alone or in combination with the forsythoside A treats cells, and all the Abeta is improved 25-35 Induced HT-22 cell damage. The mixture of the honeysuckle polysaccharide and the forsythoside A has a synergistic effectWherein the concentration ratio is 1:5, the synergy is strongest. After pretreatment of the honeysuckle polysaccharide and the forsythiaside A, the cell morphology is obviously improved, and the cell proliferation rate is obviously increased, which indicates that the honeysuckle polysaccharide and the forsythiaside A can inhibit A beta 25－35 Cytotoxicity caused.
Example 3:
the present example investigated the therapeutic effect of honeysuckle polysaccharide in combination with forsythiaside A on AD mice provided in example 1.
1. Animal feeding and grouping
Male 6 month old APP/PS1 mice and C57B6/J mice were purchased from Beijing Bei Fu Biotech Co., ltd and raised at room temperature 22℃under 12h light and dark cycle standard conditions. APP/PS1 double transgenic mice are randomly divided into 4 groups, namely a model group APP/PS1 (physiological saline 5 mL/kg/d), a honeysuckle polysaccharide group LP (200 mg/kg/d), a forsythoside A group FA (40 mg/kg/d), a honeysuckle polysaccharide combined with forsythoside A group MIX (40 mg/kg/d+200 mg/kg/d) and 6 month old CLB57 wild type mice are taken as a blank group W (physiological saline 5 mL/kg/d), and 12 mice are taken as each group. The animals were dosed by stomach irrigation for 6 weeks and body weights were measured daily.
2. Statistical analysis
Statistical software was used (version 9.0; graphPad software company, san diego, california, usa). All data are expressed as mean ± standard deviation, statistical significance was determined using one-way anova, and the confidence level of statistical significance was set at P <0.05.
3. Morris water maze
The study and memory capacity of the experimental mice was tested using the Morris water maze test. Equipment (sley WMS morris water maze system) was purchased from beijing sun instrumentation limited (beijing, china). The device comprises a black round swimming pool and an independent camera arranged on the top of the swimming pool. The diameter of the pool is 120 cm and the height is 40 cm. Before each experiment, the cells were filled with non-toxic white water (24-26 ℃ C., water depth 30 cm). Four equal quadrants are created and a white escape platform is placed in one of the quadrants. The diameter of the platform is 8 cm and the platform is positioned 1 cm below the color water surface. The experiment was performed in a quiet, temperature controlled (25 ℃) room. The experimental mice were trained on week 5, and after training for 5 days, the experimental tests were performed formally. The escape delay (i.e., the time to find the plateau) is recorded for 90 seconds.
As shown in fig. 3, the number of times of crossing the quadrant of the platform by the mice in the model group is significantly lower than that of the mice in the normal group, and the retention time in the target quadrant is also significantly reduced compared with that in the normal group. Compared with the model group, the times of crossing the quadrants of the platform by the mice of the wild group, the honeysuckle polysaccharide group and the forsythoside A group are obviously increased, and the times of crossing the quadrants of the platform and the detention time in the target quadrant by the combination of the honeysuckle polysaccharide and the forsythoside A group are obviously improved. The result shows that the honeysuckle polysaccharide and the forsythoside A can promote the formation and the consolidation of the spatial memory of the mice through the independent or combined treatment.
4. ELISA analysis of mouse serum inflammatory factor expression
To determine the expression of inflammatory cytokines, mouse serum TNF- α, IL-6 and IL-1. Beta. ELISA kits were tested according to the manufacturer's instructions (Shanghai enzyme-linked biotechnology Co., shanghai, china).
As shown in FIG. 4, the honeysuckle polysaccharide combined with the forsythoside A can reduce the protein expression of inflammatory factors including TNF-alpha and IL-6.
5. Immunofluorescence analysis of aβ protein expression in hippocampus and cortex
An important pathological feature of AD is the deposition of cortex and hippocampal amyloid aβ. The effect of forsythoside A and honeysuckle polysaccharide on APP/PS1 mouse cerebral cortex and hippocampal amyloid deposition is further observed by immunofluorescence.
APP/PS1 mice were sacrificed and brains were fixed for at least 1 day by infusion with 4% paraformaldehyde, hippocampal and cortical sections were collected and then treated with 0.1M PBS containing 0.3% Triton X-100. The above sections were fixed with 4% paraformaldehyde and then infiltrated with Triton-X100 for 30 minutes at room temperature. After blocking the non-immune serum for 30 minutes, the sections were further incubated overnight with rabbit anti-neun (1:50, affinity), GFAP (1:5000, affinity), iba1 (1:50, affinity), 8-OHdG (1:50, affinity) and GPX4 (1:50, affinity) at 4 ℃. These sections were then incubated with secondary antibodies including Alexa546 conjugated goat anti-rabbit IgG (1:50 00, siemens Feier sciences), alexa 488 conjugated goat anti-mouse IgG (1:50 00, siemens Feier sciences) and DAPI (1:1000, affinity) for 2 hours at room temperature at 37 ℃. The stained image was obtained by fluorescence microscopy (Nikon, japan) and captured by confocal laser microscopy (Lecia, germany). The number, area and fluorescence intensity of the images were quantified using Image J software.
The results are shown in fig. 5, where the white dots are amyloid, and the cortex and hippocampus of the control mice are devoid of amyloid deposition. Experimental results show that the cerebral cortex and the hippocampus of the mice in the model group are obviously changed compared with the mice in the normal group, and the sediment density and the area are increased. Compared with the model group, the number and the area of the amyloid in the brain of the mice of the honeysuckle polysaccharide group, the forsythoside A group and the honeysuckle polysaccharide and forsythoside A group are reduced to different degrees. The honeysuckle polysaccharide and forsythiaside A group and the model group have obvious differences, which shows that the honeysuckle polysaccharide and forsythiaside A group have an improving effect on amyloid plaque deposition and have recovery effects on brain injury, cognition and behavior of mice. The results indicate that the honeysuckle polysaccharide, the forsythoside A and the mixture of the honeysuckle polysaccharide and the forsythoside A can reduce the deposition of amyloid in the cortex of an APP/PS1 mouse and the sea horse, and reduce the pathological damage caused by the amyloid.
Example 4:
this example investigated the mechanism of the mixture of honeysuckle polysaccharide and forsythoside A provided in example 1 for intervention in AD based on the IL-17 signaling pathway.
1. Extraction, library construction and sequencing of brain tissue RNA
Total RNA was extracted from whole brain tissue of mice. RNA degradation and contamination were assessed in a 1% agarose gel and their concentration was determined using a qubit RNA detection kit (Life Technologies, carlsbad, CA, USA) in a qubit 2.0 fluorometer. The library was sequenced using a Illumina TruSeq RNA sample preparation kit (Illumina, san Diego, CA, USA) according to the manufacturer's recommendations, and 4 index codes were added to assign sequences to each sample. First strand cDNA synthesis uses random oligonucleotides and SuperScript II. Second strand cDNA synthesis was performed using DNA polymerase I and RNase h, with the remaining overhang converted to a blunt end by addition of exonuclease and polymerase. After the 3' -end of the DNA fragment was modified by adenylation, the Illumina PE adaptor oligonucleotide was ligated for hybridization. In 10-cycle PCR, DNA fragments with adapter molecules attached at both ends were selectively enriched using Illumina PCR primer cocktails. The index coded samples were clustered on a cluster generation system after quantitative analysis of the product using an ABI StepOnePlus real-time PCR system and an Agilent 2100 bioanalyzer system. After cluster generation, the library preparations were sequenced on the Illumina HiSeq4000 platform.
2. Gene annotation and expression profiling
After sequencing, adaptors and low quality sequences were removed using SeqPrep and sickle software and the original reads were cleaned up. Subsequently, sequence alignment was performed using TopHat2 software, the obtained clean reads were mapped to a known mouse genome (ftp:// ftp. Ensembl. Org/pub/release89/fasta/mus_musculus/dna/mus_musculus. GRCm38.Dna. Toplevel. Fa. Gz), and then assembled into genes using CuSinks software. The data provided in the supplemental material was generated using GO, KEGG, COG, NR, SWISS-PROT and PFAM databases. The expression of the annotated genes was analyzed by calculating a fragment per kilobase transcript per million map reads (FPKM) value and comparing their FPKM values using the edgeR software (version 3.8.2), thereby identifying the constant expressed genes (DEGs).
3. Verification of DEGs Using qRT-PCR and western blot
The DEGs identified by transcriptome sequencing analysis was verified using a Applied Biosystems 7900 rapid real-time PCR system (Foster, CA, USA) for quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Primers crossing exon-exon boundaries were designed for qRT-PCR using Platinum Taq polymerase (Invitrogen) and SYBR Green (Bioneer Inc., seoul, korea). And (5) carrying out data normalization treatment by using housekeeping gene beta-actin.
The whole brain tissue was homogenized in PRO-PREP protein extract (iNtRON Biotechnology inc., seengnam, korea). CAT and SREBP-1C levels were determined using 20. Mu.g of holoprotein. Proteins were gel-electrophoretically separated on a 10% Tris-HCl polyacrylamide gel according to the molecular weight of the protein, and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% BSA in TBST, then incubated with specific primary antibodies (diluted in TBST, 1:1000) overnight at 4"C, then incubated with the corresponding enzyme-linked anti-rabbit/mouse secondary antibodies (diluted in TBST, 1:500) for 1 hour at room temperature. ECL selection Western Blotting detection Regent kit (GE Healthcare, chicago, IL, USA) was used in the protein band detection process. Membranes were scanned using a Bio-Rad Chemidoc XRS imaging system and band intensities were analyzed using Bio-Rad quality One software (Bio-Rad, calif., USA).
4. Results and analysis
In this example, a mouse brain tissue cDNA library was constructed using an Illumina Hiseq4000 sequencing platform and sequenced to evaluate the mouse brain gene expression profile. The analysis obtains 252.76Gb Clean Data altogether, each sample Clean Data reaches more than 6.9Gb, and the percentage of Q30 base is more than 92.94%; respectively carrying out sequence alignment on clear Reads of each sample and a designated reference genome, wherein the alignment rate is different from 95.81% to 96.55%; analysis of expression level: the analysis detects 34130 expressed genes in total, wherein 33639 known genes and 491 new genes; a total of 122410 transcripts were expressed, of which 104511 transcripts were known and 17899 new transcripts. In addition, clear reads with unique and multiple positions on the reference genome were obtained, and can be successfully aligned to both positive and negative clear reads of the reference genome, indicating that this transcriptome data was valid for further analysis.
Based on the quantitative result of the expression level, the differential gene expression of the transcriptomes of different treatment groups was compared. Performing inter-group differential gene analysis to obtain genes with differential expression between two groups, wherein the differential analysis software comprises the following steps: DESeq2, screening threshold is: log2fc| > = 1& p value <0.05, the results indicate that 449 genes in W group are differentially expressed (more than 1.5 fold, p value < 0.05) from APP/PS1 group, of which 388 are up-regulated and 111 are down-regulated; there were 419 genes in the MIX and there was differential expression from the APP/PS1 group (139 genes up-regulated and 280 genes down-regulated, respectively). To evaluate the mechanism of influence of honeysuckle polysaccharide in combination with forsythoside a on AD pathology, 419 differentially expressed genes of MIX and APP/PS1 groups were analyzed using DAVID database, as shown in fig. 6, 139 up-regulated and 280 down-regulated differentially expressed genes were mainly enriched in "signal molecular reaction", "immune system" and "signal transduction", indicating that honeysuckle polysaccharide in combination with forsythoside a might improve AD mouse defects by participating in these pathways.
As shown in FIG. 7, the analysis result of transcriptome data shows that the mRNA expression level of the gene Il-17a in the brain tissue of a model group (APP/PS 1) is significantly higher than that of a wild group (W) mouse, and the mRNA expression level of the gene Il-17a in the brain tissue of a honeysuckle polysaccharide and forsythiaside A administration group (MIX) mouse is significantly lower than that of the gene APP/PS1, and also the mRNA expression level of the gene Act1 in the brain tissue of the model group mouse is significantly higher than that of the wild group (W), and the mRNA expression level of the gene Act1 in the brain tissue of the honeysuckle polysaccharide and forsythiaside A administration group (MIX) is significantly lower than that of the model group; regarding the Nfkbp65 gene, the analysis result shows that the mRNA level of the gene in the model group (APP/PS 1) is obviously up-regulated compared with that in the wild group (W), and the mRNA expression level of the gene in the honeysuckle polysaccharide and forsythiaside A administration group is obviously down-regulated compared with that in the model group. Regarding the Cox2 gene, the analysis result shows that the mRNA level of the gene in the model group (APP/PS 1) is obviously up-regulated compared with that in the wild group (W), and the mRNA expression level of the gene in the honeysuckle polysaccharide and forsythiaside A administration group is obviously down-regulated compared with that in the model group.
This portion of transcriptome data was validated using qRT-PCR and the results were consistent with mRNA sequencing data, i.e., relative expression was reduced in the honeysuckle polysaccharide in combination with forsythoside a-dosed (MIX) mice Il-17a, act1, ifkba, nfkbp65, and Cox2 compared to model group (APP/PS 1) mice. Western blot verification shows that the honeysuckle polysaccharide and the forsythiaside A can inhibit the increase of the expression quantity of IL-17A and Nfkbp65 proteins. These results indicate that honeysuckle polysaccharide in combination with forsythoside A can alleviate neuroinflammation in AD pathology by regulating the expression of genes related to IL-17 signaling pathway.
Example 5:
this example investigated the mechanism of intervention of the mixture of honeysuckle polysaccharide and forsythoside a provided in example 1 into AD based on intestinal flora.
To avoid the influence of environmental and dietary factors on the intestinal flora, all experimental animals were fed with the same diet in the same feeding room.
1. Materials and methods
The intestinal tract content tissue is taken from colon, frozen by liquid nitrogen and stored at-80 ℃ for standby. Genomic DNA was extracted using a DNA extraction kit, 5 stool samples per group. The diversity detection of intestinal flora of AD mice is carried out by adopting a 16S rRNA sequencing method.
The purity and concentration of the DNA were determined by agarose gel electrophoresis. The sample was diluted to 1 ng/. Mu.l-1 with sterile water. The diluted genomic DNA was used as a template and the 16S V3-V4 region was used as a sequencing region. Specific barcode primers were used. Primers 343F (5 '-TacgGraggCAGcG-3') and 798R (5'-AgggtatCtaatCCT-3') were used for PCR using Takara Ex Taq high fidelity enzyme (Bio-Rad, CA, USA). The PCR product is subjected to electrophoresis detection and magnetic bead purification, and is used as a template for double-round PCR amplification. After purification, the qubits are quantized.
Finally, aliquots were mixed according to PCR product concentration and sequenced on an Illumina-Miseq (Cal, USA) machine. The original two-terminal sequence (FASTQ format) is sorted using trigonometric software. When the quality is less than 20, the previous high quality sequence is intercepted. After the high quality sequences are generated, they are classified into a plurality of OTUs according to the similarity of the sequences using Vsearch software. Sequence similarity above 97% is categorized as one OTU unit. Representative sequences for each OTU were screened using the QIIME software package. All representative sequences were compared and annotated with the Silva (Version 132) database, retaining annotated results with confidence intervals greater than 0.7.
2. Results and analysis
The Kruskal-Wallis test was used to analyze whether the inter-group Alpha diversity index differences were significant. Taking the case of the Observe, chao1 and Shannon indices, box plots of the inter-group variance analysis are shown in fig. 8. For the gene model mice, the Observe and Chao1 indexes show that the gene model mice have obvious differences from the control group, which indicates that the intestinal flora of the gene model mice is obviously disturbed. After the honeysuckle polysaccharide is combined with the forsythoside A, the normal intestinal flora of a model mouse is obviously affected.
As shown in fig. 9, forsythoside a administration in combination with honeysuckle polysaccharide had a significant effect on abundance on the gut flora phylum category. AD model mice had a significant change in the abundance of intestinal flora over the phylum category compared to wild-group mice. The model mice had a decrease in firmicutes door abundance, while the bacteroides door abundance increased. The forsythoside A and the honeysuckle polysaccharide can be combined for administration to correct the abnormality of the intestinal flora of the AD model in the abundance of the portal class. For the gene model mice, the effect of the relative proportion of the administration reversion of the phylum thick-walled bacteria and the phylum bacteroides is more obvious. These results indicate that forsythoside a administration in combination with honeysuckle polysaccharide has a significant effect on intestinal flora. The phylum Thick-walled bacteria are generally considered to be beneficial bacteria, and the phylum bacteroides are generally considered to be harmful bacteria. The application of forsythoside A combined with the honeysuckle polysaccharide can obviously improve the proportion of the genome mouse firmicutes and the bacteroides, promote the increase of beneficial bacteria, and indicate that the possible disturbance of intestinal flora can be corrected by the forsythoside A combined with the honeysuckle polysaccharide.
By analyzing the intestinal microbiota levels of the three groups of mice, it was also found that the relative abundance was significantly reduced in AD group compared to WT group trichomonad, while the abundance was significantly increased after forsythoside a combined with the intervention of the honeysuckle polysaccharide (fig. 9). 16S rRNA sequencing is limited by read length and amplified region, and at most, relatively accurate flora information can be obtained at the genus level. Therefore, in the comparison of the effect of further regulating intestinal flora by combining forsythoside and honeysuckle polysaccharide, 16S sequencing minimum resolution level, namely the genus level is continuously selected, and the inter-group bacteria difference condition is observed. The results show that the abundance of Muribaculum is significantly reduced in the model group, while the combination group significantly increases the abundance of Muribaculum, which is confirmed to be closely related to inflammatory response. In addition, bacteroides are significantly reduced in the dosing group, and the bacteria abundance can contribute to bacteroides abundance in the model group, thereby leading to a maladjustment in the ratio of bacteroides to bacteroides, which has an important link to the occurrence of neurological diseases. It can be deduced from the above results that administration of honeysuckle polysaccharide in combination with forsythoside a may improve cognitive dysfunction in AD model mice by modulating the abundance of specific species.
The honeysuckle polysaccharide and forsythiaside A combined provided by the invention can improve cognitive dysfunction of an APP/PS-1 double-transgenic AD model mouse, can regulate the composition, structure and abundance of specific species of intestinal flora, and can also relieve intestinal microbial dysfunction caused by AD to a certain extent. Therefore, the honeysuckle polysaccharide and the forsythiaside A can relieve AD symptoms by adjusting the structure, the composition and the function of intestinal flora.
The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.
Claims (9)
1. A traditional Chinese medicine extract for improving Alzheimer disease is characterized by comprising honeysuckle polysaccharide and forsythoside A; the mass concentration ratio of the forsythoside A to the honeysuckle polysaccharide is 1:2 to 1:6; the honeysuckle polysaccharide is prepared by the following steps: pulverizing flos Lonicerae, extracting with ultrasonic water extraction method, centrifuging to obtain supernatant, concentrating, adding ethanol, standing, centrifuging to obtain precipitate, dissolving the precipitate with water, extracting to remove protein to obtain crude polysaccharide of flos Lonicerae; purifying the honeysuckle crude polysaccharide by column chromatography to obtain the honeysuckle polysaccharide.
2. The traditional Chinese medicine extract for improving alzheimer's disease according to claim 1, characterized in that: the extraction condition of the ultrasonic water extraction method is that the ultrasonic time is 25-35min, the ultrasonic temperature is 55-65 ℃, and the ratio of feed to liquid is 1:25-1:35 in terms of volume-mass ratio.
3. The traditional Chinese medicine extract for improving alzheimer's disease according to claim 1, characterized in that: adding a Savag reagent for extraction deproteinization; the Savag reagent comprises chloroform and n-butanol; the volume ratio of the chloroform to the n-butanol is 4:1.
4. the traditional Chinese medicine extract for improving alzheimer's disease according to claim 1, characterized in that: purifying the honeysuckle crude polysaccharide by using a DEAE-52 cellulose chromatographic column and a Sephadex G-100 gel chromatographic column to obtain the honeysuckle polysaccharide.
5. The traditional Chinese medicine extract for improving alzheimer's disease according to claim 1, wherein the preparation method of forsythoside a is as follows: pulverizing folium forsythiae, placing in a container, leaching with ethanol solution, adsorbing with resin, eluting, and treating the eluate with reversed phase chromatography to obtain forsythoside A.
6. The traditional Chinese medicine extract for improving alzheimer's disease according to claim 5, characterized in that: the concentration of ethanol in the leaching process is 40-50%, and the liquid-material ratio is 25:1-30 in terms of volume-mass ratio: 1, the extraction temperature is 40-60 ℃ and the extraction time is 20-30min.
7. The traditional Chinese medicine extract for improving alzheimer's disease according to claim 5, characterized in that: in the adsorption and elution processes, AB-8 macroporous adsorption resin is used as an adsorbent, 5-10BV of water is used for eluting, then 5-10BV of 30% ethanol solution and 5-10BV of 50% ethanol solution are used for eluting respectively, and the eluting flow rate is 2-5BV/h.
8. Use of a traditional Chinese medicine extract according to any one of claims 1-7 for the preparation of a medicament for improving alzheimer's disease.
9. The use of the extract of chinese medicine of claim 8 for the preparation of a medicament for ameliorating alzheimer's disease, characterized in that: the medicine takes forsythoside A and honeysuckle polysaccharide as active ingredients and also comprises pharmaceutically acceptable auxiliary materials.
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| US6083921A (en) * | 1998-01-12 | 2000-07-04 | Xu; Kai Jian | Pharmaceutical compositions and method of using same |
| CN104177510A (en) * | 2013-05-21 | 2014-12-03 | 中国科学院上海药物研究所 | Alpha-1,4-glucan and its preparation method and use |
| CN111001181A (en) * | 2019-11-29 | 2020-04-14 | 皖南医学院 | Honeysuckle extraction method with high polysaccharide preservation rate and ultralow-temperature ultrafine pulverizer |
| CN115304687A (en) * | 2022-09-01 | 2022-11-08 | 北京中医药大学 | Flos lonicerae polysaccharide for improving non-alcoholic fatty liver disease and preparation method and application thereof |
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| US6083921A (en) * | 1998-01-12 | 2000-07-04 | Xu; Kai Jian | Pharmaceutical compositions and method of using same |
| CN104177510A (en) * | 2013-05-21 | 2014-12-03 | 中国科学院上海药物研究所 | Alpha-1,4-glucan and its preparation method and use |
| CN111001181A (en) * | 2019-11-29 | 2020-04-14 | 皖南医学院 | Honeysuckle extraction method with high polysaccharide preservation rate and ultralow-temperature ultrafine pulverizer |
| CN115304687A (en) * | 2022-09-01 | 2022-11-08 | 北京中医药大学 | Flos lonicerae polysaccharide for improving non-alcoholic fatty liver disease and preparation method and application thereof |
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