CN105663324B - Effective part group of yang tonifying and five returning decoction and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of medicines, relates to application of a plurality of traditional Chinese medicine mixed compounds, and particularly relates to application of an effective part group of Buyang Huanwu decoction in inducing differentiation of mesenchymal stem cells of bone marrow to nerve cells. The effective components include glycosides, total alkaloids, radix Angelicae sinensis polysaccharide, safflower yellow and naringenin. Research shows that the effective part group of the yang tonifying and five returning decoction can induce the mesenchymal stem cells of rats to differentiate into neuron-like cells in vitro, and the effective part group can be applied to medicines for treating nervous system diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to application of a plurality of traditional Chinese medicine mixed compounds, and particularly relates to an effective site group of Buyang Huanwu decoction and application thereof in inducing mesenchymal stem cells of bone marrow to differentiate towards nerve cells.
Background
Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic cells derived from bone marrow tissues, and have the advantages of easily obtained sources, no ethical limitation, wide proliferation and differentiation capacity, easy separation, culture, amplification and purification, weak immunogenicity, strong immune tolerance, high in vitro gene transfection rate, capability of stably and efficiently expressing exogenous genes and the like. Thus, MSCs become a relatively ideal seed cell. In recent years, it has been reported that MSCs can differentiate into neuron-like cells in vivo, and rat and human BMSCs can be induced into neuron-like cells in vitro using basic fibroblast growth factor (bFGF), dimercaptoethanol (BME), dimethyl sulfoxide (DMSO), Butylated Hydroxyanisole (BHA), Epidermal Growth Factor (EGF), Retinoic Acid (RA), and the like as an inducer. The MSCs are convenient to obtain and easy to obtain from the self body, and immune rejection reaction cannot occur after replanting; easy to separate, culture and amplify in vitro; high transfection efficiency in vitro and can stably and efficiently express exogenous genes, thereby being an ideal seed cell. This provides a new concept for the treatment of nervous system injuries and degenerative diseases. The chemical synthetics such as DMSO, B-mercaptoethanol and the like have strong nerve cell induction effect on MSCs, but the obvious toxicity, teratogenicity and carcinogenicity limit the application. The traditional Chinese medicine is based on thousands of years of clinical experience, and has relatively small toxic and side effects.
The traditional Chinese medicine formula, namely yang-tonifying and five-returning decoction, is clinically used for treating the ischemic stroke sequelae, and the modern pharmacological research shows that the traditional Chinese medicine formula has the effects of regulating immunity, resisting inflammation, regulating lipid metabolism, dilating cerebral vessels, improving microcirculation, improving hemorheology indexes, resisting blood coagulation, inhibiting thrombosis, resisting free radicals, protecting nerves and the like. When the MSCs are transplanted in the animal body, the survival rate, the homing rate and the differentiation rate of the MSCs can be improved by the intervention of the Yang tonifying and five returning decoction. However, the traditional Chinese medicine has complex ingredients, and has the characteristics of multiple causes, multiple effects and multiple targets when playing the effect.
Disclosure of Invention
The invention aims to provide an effective part group of yang tonifying and five returning decoction and application thereof in inducing mesenchymal stem cells of bone marrow to differentiate towards nerve cells, and the effective part group of yang tonifying and five returning decoction can be applied to medicines for treating nervous system diseases. The effective fraction can promote differentiation of bone marrow mesenchymal stem cells and induce differentiation of bone marrow mesenchymal stem cells into neuron-like cells.
Since the background art describes that the traditional Chinese medicine formula of yang-tonifying and five-reducing decoction is clinically used for treating the sequelae of ischemic stroke, modern pharmacological studies find that the traditional Chinese medicine formula of yang-tonifying and five-reducing decoction has the effects of regulating immunity, resisting inflammation, regulating lipid metabolism, dilating cerebral vessels, improving microcirculation, improving hemorheological indexes, resisting blood coagulation, inhibiting thrombosis, resisting free radicals, protecting nerves and the like. Through years of research and further experiments of the inventor, the inventor finds that in the previous stage, the LD50 of each component in the yang tonifying and five returning decoction is searched by using a sequential method, the optimal compatibility dosage is measured by using an orthogonal design method, each effective part group (active principal region of Buyang Huang decoction apr-BYHD) is extracted from the yang tonifying and five returning decoction formula, and the apr-BYHD can induce the rat bone marrow stem cells to be differentiated into neuron-like cells in vitro and has an inducing effect superior to that of the yang tonifying and five returning decoction formula, so the inventor conjects that the effective part group can be applied to the medicines for treating nervous system diseases.
The invention provides an application of effective parts of yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells, wherein the effective parts of the yang tonifying and five returning decoction are as follows: glycoside compounds, total alkaloids, angelica polysaccharide, safflower yellow and naringenin; the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: extracting radix astragali, radix Paeoniae Rubra and Lumbricus with ethanol under reflux, and filtering to obtain residue A; recovering ethanol from the filtrate, adjusting to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adsorbing the concentrated solution with macroporous resin column, washing with water, eluting with ethanol, collecting ethanol eluate, concentrating, and drying to obtain glycosides and naringenin effective components;
(2) separating and extracting the effective part of the safflower yellow: decocting semen Persicae and Carthami flos in water, filtering, mixing filtrates, concentrating to obtain concentrated solution with concentration of 0.5-3g medicinal materials per 1ml, adsorbing with macroporous resin column, washing with water, eluting with ethanol, collecting eluate, concentrating, and drying to obtain safflower yellow effective component;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in water, filtering to obtain filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 45-60%, stirring, standing, filtering to obtain precipitate B, volatilizing ethanol from the filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 80-85%, stirring, standing, collecting precipitate, drying to obtain effective fraction of Angelica polysaccharide, collecting ethanol precipitation solution, volatilizing ethanol, adding precipitate B for dissolving, adding water to adjust to concentrate solution of 0.5-3g medicinal material per 1ml, adsorbing with D101 macroporous resin column, washing with water, eluting with ethanol, collecting eluate, concentrating, and drying to obtain effective fraction of total alkaloids.
The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells comprises the following steps: glycoside compounds, total alkaloids, angelica polysaccharide, safflower yellow and naringenin; the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: percolating radix astragali, radix Paeoniae Rubra and Lumbricus with ethanol, collecting percolate, and collecting residue A; recovering ethanol from percolate, adjusting to concentrate of 0.5-20g medicinal materials per 1ml, adding the concentrate to the top of polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, collecting 100ml eluate as a fraction, monitoring dynamic change of components of the eluted fraction by high performance liquid chromatography, mixing fractions with similar components as a component, collecting fractions of effective parts of astragaloside, paeoniflorin and naringenin, concentrating, and drying to obtain glycosides and naringenin effective parts;
(2) separating and extracting the effective parts of the safflor yellow and the naringenin: decocting semen Persicae and Carthami flos in water, filtering, mixing filtrates, concentrating to 1ml concentrated solution equivalent to 0.5-3g medicinal materials, adding the concentrated solution to the top of polyamide column, sequentially eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol, collecting 100ml eluate as a fraction, monitoring dynamic change of eluted fraction components by high performance liquid chromatography, collecting fractions of safflor yellow and naringenin, concentrating, and drying to obtain effective fractions of safflor yellow and naringenin;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in water, filtering, mixing filtrates, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 45-60%, stirring, standing, filtering to obtain precipitate B, volatilizing ethanol from filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 80-85%, stirring, standing, collecting precipitate, and drying to obtain polysaccharide effective component; taking the alcohol precipitation solution, volatilizing ethanol, adding the precipitate B for dissolution, adding water for adjusting to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adding the concentrated solution to the top of a polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, taking 100ml of eluent as a fraction, monitoring dynamic change of components of the eluted fraction by a high performance liquid chromatography, combining fractions with similar components as a component, collecting fractions of effective parts of ligustrazine and ferulic acid, concentrating and drying to obtain the effective parts of the total alkaloids.
The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate into nerve cells is disclosed, wherein the types and the contents of the medicinal materials used for preparing the effective part group of the yang tonifying and five returning decoction are as follows: 23-66 g of astragalus root, 6-17 g of red peony root, 6-10g of ligusticum wallichii, 9-19 g of angelica, 9-17 g of earthworm, 6-16 g of safflower and 9-20 g of peach kernel.
The application of the effective site group of the Yang tonifying and Wu Tang is used for inducing the differentiation of the bone marrow mesenchymal stem cells to the nerve cells, and is characterized in that the application of the effective site group of the Yang tonifying and Wu Tang is used for treating the nervous system diseases, the application of the effective site group of the Yang tonifying and Wu Tang is used for preparing the medicine for promoting the transformation of the bone marrow mesenchymal stem cells from the body to the direction of the neuron-like cells, and the bone marrow mesenchymal stem cells are directionally differentiated into the neuron-like cells.
The MEK-ERK and p38MAPK signal channels participate in the process that the effective parts of the Yang recuperating decoction induce the BMSCs to directionally differentiate towards nerve cells in the process that the mesenchymal stem cells of the marrow are directionally differentiated into neuron-like cells, and the blockage of the MEK-ERK and p38MAPK signal channels can inhibit the nerve differentiation of the BMSCs induced by the combination of the effective part formulas of the traditional Chinese medicines.
The invention also aims to solve the technical problem of providing a traditional Chinese medicine effective part group capable of promoting the in vitro directional transformation of the mesenchymal stem cells into neuron-like cells. Therefore, the technical scheme adopted by the invention is as follows: the effective part group of the Yang tonifying and Wu Heng decoction is used as a cell differentiation promoting agent to induce the bone marrow mesenchymal stem cells to directionally differentiate into neuron-like cells.
The effective part group of the yang tonifying and five returning decoction provided by the invention can promote the differentiation of the mesenchymal stem cells and induce the differentiation of the mesenchymal stem cells into neuron-like cells, is prepared into a medicament for treating nervous system diseases, and develops a new cell replacement therapy for various refractory diseases of the nervous system.
Drawings
FIG. 1 shows primary and primary cells in subculture (. times.100) of BMSCs;
FIG. 2 shows primary and passage 2 cells in primary and subculture (. times.100) of BMSCs;
FIG. 3 shows primary and passage 3 cells in primary and subculture (. times.100) of BMSCs;
FIG. 4 is a cell diagram of a flow cytometry blank for detecting rat BMSCs cells;
FIG. 5 is a diagram of flow cytometry to detect BMSCs positively expressed by FITC-labeled CD90 in rat BMSCs cells;
FIG. 6 is a diagram of flow cytometry to detect BMSCs positively expressed by FITC-labeled CD11 in rat BMSCs cells;
FIG. 7 is a diagram of flow cytometry to detect BMSCs positively expressed by FITC-labeled CD45 in rat BMSCs cells;
FIG. 8 shows the Western-Blots detection of p-ERK protein expression electropherograms of BMSCs induced by Yang tonifying and Wu Fang decoction (1 blank control cell, 2 negative control group, 3 apr-BYHHD group);
FIG. 9 shows the Western-Blots detection of p-p38 protein expression electropherograms of BMSCs induced by Yang tonifying and Wu Fang decoction (1 blank control cell, 2 negative control group, 3 apr-BYHDD group);
FIG. 10 is a gel electrophoresis pattern of mRNA expression of BMSCs NSE induced after detection of MAPK pathway blockade by RT-PCR (1: apr-BYHDD control group; 2: apr-BYHDD + SB203580 group; 3: apr-BYHDD + PD98059 group; M: Marker);
FIG. 11 is a gel electrophoresis pattern of the expression of BMSCs NSE protein induced after MAPK pathway blockade detected by Western-Blots (1: apr-BYHHD control group; 2: apr-BYHHD + SB203580 group; 3: apr-BYHHD + PD98059 group);
FIG. 12 is a gel electrophoresis of the expression of BMSCs Nestin mRNA induced by RT-PCR detection of MAPK pathway blockade (1: apr-BYHDD control group; 2: apr-BYHDD + SB203580 group; 3: apr-BYHDD + PD98059 group; M: Marker);
FIG. 13 is a gel electrophoresis pattern of expression of BMSCs Nestin protein induced after MAPK pathway blockade detected by Western-Blots (1: apr-BYHDD control group; 2: apr-BYHDD + SB203580 group; 3: apr-BYHDD + PD98059 group).
Detailed Description
The research of the embodiment shows that the effective part combination of the yang tonifying and five returning decoction can promote the directional differentiation of the mesenchymal stem cells of the bone marrow into the neuron-like cells in vitro. The invention is further described by combining the drawings and the embodiments in the specification.
Example 1
The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells comprises the following steps: glycoside compounds, total alkaloids, angelica polysaccharide, safflower yellow and naringenin; the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: taking 60g of astragalus, 6g of red paeony root and 9g of earthworm, adding 5-15 times of 40% -70% ethanol, carrying out reflux extraction for 1-3 times, each time for 1-2h, and filtering to obtain medicine residue A for later use; recovering ethanol from the filtrate, adjusting to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adsorbing the concentrated solution with macroporous resin column, washing with water, eluting with 40-50% ethanol, collecting ethanol eluate, volatilizing ethanol, concentrating, and drying to obtain glycosides and naringenin effective components;
(2) separating and extracting the effective part of the safflower yellow: taking 9g of peach kernels and 9g of safflower, adding 5-20 times of water, decocting for 1-3 times, each time for 1-2 hours, filtering, merging the filtrates, concentrating to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adsorbing the concentrated solution by a macroporous resin column, washing with water, eluting with 20-30% ethanol, collecting the eluent, volatilizing the ethanol, concentrating, drying to obtain the carthamus yellow effective part;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, 9g radix Angelicae sinensis and 6g rhizoma Ligustici Chuanxiong with 5-20 times of water for 1-3 times, each for 1-2 hr, filtering, mixing filtrates, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 45-60%, stirring, standing for 12-24 hr, filtering to obtain precipitate B, volatilizing ethanol from filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 80-85%, stirring, standing for 12-24 hr, collecting precipitate, vacuum drying to obtain effective fraction of Angelica polysaccharide, collecting ethanol precipitation solution, volatilizing ethanol, adding precipitate B for dissolving, adding water to adjust to concentrate solution with relative density of 0.5-3g medicinal materials per 1ml, adsorbing with D101 macroporous resin column, washing with water, eluting with 60-70% ethanol, collecting eluate, volatilizing ethanol, concentrating, and drying to obtain effective components of total alkaloids.
Example 2
The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells comprises the following steps: glycoside compounds, total alkaloids, angelica polysaccharide, safflower yellow and naringenin; the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: taking 60g of astragalus, 6g of red paeony root and 9g of earthworm, adding 5-15 times of 40-70% ethanol, performing percolation extraction, and collecting percolate, wherein medicine residue A is reserved; recovering ethanol from percolate, adjusting to concentrate of 0.5-20g medicinal materials per 1ml, adding the concentrate to the top of polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, collecting 100ml eluate as a fraction, monitoring dynamic change of components of the eluted fraction by high performance liquid chromatography, mixing fractions with similar components as a component, collecting fractions of effective parts of astragaloside, paeoniflorin and naringenin, concentrating, and drying to obtain glycosides and naringenin effective parts;
(2) separating and extracting the effective parts of the safflor yellow and the naringenin: taking 9g of peach kernels and 9g of safflower, adding 5-20 times of water, decocting for 1-3 times, each time for 1-2 hours, filtering, merging the filtrates, concentrating to obtain concentrated solution with each 1ml being equivalent to 0.5-3g of medicinal materials, taking the concentrated solution to add on the top of a polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, taking 100ml of eluent as a fraction, monitoring the dynamic change of the components of the eluted fraction by a high performance liquid chromatography, collecting fractions of yellow pigment and naringenin, concentrating and drying to obtain the effective parts of safflor yellow and naringenin;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, 9g of radix Angelicae sinensis and 6g of rhizoma Ligustici Chuanxiong with 5-20 times of water for 1-3 times, each for 1-2 hr, filtering, mixing filtrates, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to reach ethanol content of 45-60%, stirring, standing for 12-24 hr, filtering to obtain precipitate B, removing ethanol from filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to reach ethanol content of 80-85%, stirring, standing for 12-24 hr, collecting precipitate, and drying to obtain Angelica polysaccharide effective component; taking the alcohol precipitation solution, volatilizing ethanol, adding the precipitate B for dissolution, adding water for adjusting to each 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adding the concentrated solution to the top of a polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, taking 100ml of eluent as a fraction, monitoring dynamic change of components of the eluted fraction by a high performance liquid chromatography, merging fractions with similar components as a component, collecting fractions of effective parts of ligustrazine and ferulic acid, volatilizing ethanol, concentrating and drying to obtain the effective parts of the total alkaloids.
Example 3
The effective part group of the yang tonifying and five returning decoction is used for promoting the induced differentiation experiment research of the bone marrow mesenchymal stem cells directionally differentiating into neuron-like cells in vitro and the effect analysis of MAPK in the induced differentiation experiment research.
Materials:
animals: 10 male SD rats of 6-8 weeks old, clean grade, body mass (130 + -20) g, purchased from Shanghai Si Laike laboratory animals, Inc., animal license number: SCXK (Shanghai) 2009-0004. The temperature and humidity of the animal room are 25 ℃ and 60 percent respectively. The operation of the animals in the rat experiments is carried out according to the relevant regulations in the guidance comments on the animals to be tested issued by the Ministry of science and technology of the people's republic of China.
Medicine preparation: astragalus (latin scientific name:Leguminosae) 60g, red peony root (Latin school name: paeonia lactiflorpall.) 6g, Ligusticum wallichii (Latin scientific name: rhizoma Chuanxiong) 6g, Chinese Angelica (academic name: Angelica sinensis)9g, earthworm [ Latin academic name Pheretima (E.Perrier)) 9g, peach kernel [ Latin academic name: prunuspersica (L.) Batsch)]9g and safflower (Latin name: Carthamus tinctorius L.) 9 g. According to the composition ratio, the effective part group is prepared by the method in the embodiment 1 or the embodiment 2 (the original extraction liquid is prepared by a water-decoction alcohol precipitation method, then the effective parts are extracted from the original extraction liquid by an acid-base precipitation and ion exchange resin chromatography method, the effective parts are completely separated, the effective parts are not mixed with each other, the purity is more than 70 percent, the content of a quality control substance is determined by a high pressure liquid chromatography and a chemical analysis method according to the following standards, wherein the content of ligustrazine in alkaloid is 9.76mg/g alkaloid), and the required concentration is prepared by a serum-free L-DMEM culture medium.
The method comprises the following steps:
(1) isolation, culture and identification of BMSCs: male SD rats are sacrificed by cervical dislocation and soaked in 75% ethanol for 10 min. The tibia and femur were quickly removed under sterile conditions and the ends were resected. The bone marrow cavity was repeatedly washed with DMEM solution aspirated by a No. 8 needle, and the cells were collected. Adding DMEM medium (GIBCO, USA) containing 10% fetal bovine serum (Hyclone, USA), repeatedly beating, counting cells, and adding 1 × 109seed/mL at 50cm basal area2In a culture flask, the content of CO with the volume fraction of 0.05 at the temperature of 37 ℃ and the saturation humidity2Incubators (THERMO FORMA, USA). After 48h the suspension was discarded and the solution was changed for the first time. Following 1 exchange every 2d, suspension-grown hematopoietic cells were discarded with the exchange, leaving behind adherently-grown BMSCs. Approximately after the cells fused to approximately 80% at the bottom of the flask, the cells were harvested after digestion with 0.25% trypsin (SIGMA, USA). Repeating the above operations until reaching the No. P3And (4) generation. Taking 3 rd generation BMSCs, digesting with 0.25% pancreatin to obtain cell suspension, adding 1% BSA blocking solution for 10min, washing with PBS for 3 times, adding FITC fluorescent labeled anti-CD 11b, CD45, CD90 anti-CD 110.5mL (SERATEC, Germany) of the antibody (1: 1000), PBS was added to the control group, the control group was incubated at 4 ℃ for 30min, centrifuged at 1500r/min for 5min, washed 3 times with PBS, fixed with 1% paraformaldehyde, and examined for cell surface antigens using a FACScan flow cytometer (BD, USA). The cells obtained are identified as high-purity BMSCs by CD45, the CD11b positive rate is lower than 5%, and the CD90 positive rate is higher than 95%.
(2) Expression of phosphoproteins ERK, p-38 in BMSCs following apr-BYHDD induction:
experiments were performed with BMSCs passed to passage 4. The experiment was divided into 3 groups.
① Experimental Observation group, rat BMSCs culture medium added with 0.039g/L apr-BYHDD.
② negative control group rat BMSCs were cultured in DMEM equivalent to ① groups.
③ Normal control group rat BMSCs was cultured in DMEM containing 10% fetal bovine serum in an amount equivalent to ① groups.
And after 3 groups of the cells are all placed in an incubator to be incubated for 24 hours, removing the culture solution of each group, washing the cells for 2 times by using a DMEM (DMEM) culture medium, and continuously culturing the cells according to the culture conditions, wherein the culture solution is changed for 1 time every 3 days. After induced differentiation for 7 days, 14 days and 28 days, BMSCs of each group are respectively taken out and identified as phosphorylated proteins ERK and p-38.
(3) Effect of MAPK signaling pathway on apr-BYHWD induction of neural differentiation of BMSCs:
BMSCs passed to passage 4 were randomly assigned to apr-BYHDD control, apr-BYHDD + PD98059, and apr-BYHDD + SB 203580.
① apr-BYHHD control group, 3 ml of induction culture medium containing apr-BYHHD (0.39%) was added to culture BMSCs for 24h, the culture medium was removed, the BMSCs were washed with DMEM medium for 2 times, and the BMSCs were cultured under the above-mentioned conditions, and the culture medium was changed 1 time every 3d, induced differentiation was carried out for 7 days, 14 days, and 28 days, and then, neural-like cells were identified.
② apr-BYHWD + PD98059 group BMSCs were cultured by adding 3 ml of induction medium containing apr-BYHWD (0.39%), adding 30. mu.l of 1000. mu. mol/L PD98059(MEK-ERK signaling pathway inhibitor, SIGMA corporation, USA) together with the apr-BYHDD induction solution, diluting PD98059 to 10. mu. mol/L, incubating for 24 hours, removing the culture medium, washing with DMEM medium for 2 times, culturing under the above culture conditions, changing the medium 1 time every 3 days, inducing differentiation for 7 days, 14 days, and 28 days, and identifying neural cells.
③ apr-BYHHD + SB203580 group BMSCs were cultured by adding 3 ml of induction medium containing apr-BYHHD (0.39%) and then adding 30. mu.l of 1500. mu. mol/L SB203580(p38 MAPK signal transduction pathway inhibitor, SIGMA corporation, USA) together with the apr-BYHDD induction solution to dilute SB203580 to 15. mu. mol/L, after incubating for 24 hours, the culture medium was removed and washed with DMEM medium 2 times, and then the culture was continued under the above culture conditions, and the culture medium was changed 1 time every 3 days and induced differentiation was carried out for 7 days, 14 days, and 28 days, followed by neural-like cell identification.
(4) RT-PCR detection of cellular Nestin and NSE mRNA expression:
BMSCs at different time intervals after induction were digested with 0.25% pancreatic enzyme and prepared into cell suspensions of approximately 1X 107Cell lysis and extraction of total cellular RNA were performed by Trizol (Invitrogen, Canada). After the integrity of the gel was identified by electrophoresis, the integrity of the gel was measured by using a nucleic acid protein analyzer of DU640 type (Backman, Inc., USA)260And A280The Primer sequence published by NCBI Genbank was designed and Blast verified using Primer Premier5.0 (Premier Biosoft, USA), and synthesized by Invitrogen.
PCR primer sequence and amplified target fragment:
RT-PCR was performed in two steps, using a gradient PCR apparatus (Bio-rad, USA) to synthesize cDNA at 65 deg.C, 5min, 42 deg.C, 60min, followed by 70 deg.C denaturation for 5min, followed by 35 cycles of PCR amplification (Nestin: 94 deg.C, 30s, 54 deg.C, 1min, 72 deg.C, 1 min; NSE: 94 deg.C, 30s, 56 deg.C, 1min, 72 deg.C, 1 min), and finally extension at 72 deg.C for 10min, after PCR was completed, amplification was detected by 2% agarose gel electrophoresis, and the absorbance ratios of Nestin, NSE mRNA and internal reference β -actin mRNA were analyzed by scanning using a gel imaging system (UVItec, UK), and the experiment was repeated 3 times.
(5) And (3) detecting contents of phosphorylated ERK, phosphorylated p-38, Nestin and NSE proteins by Western-blots:
BMSCs induced for different periods of time were extracted for total protein, BCA was used to determine protein concentration, SDS-PAGE was run and transferred to membrane, PVDF after electrotransfer was placed to dilute rabbit anti-rat p-ERK, p-p-38, Nestin and NSE monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) to appropriate concentrations with TBST at 1:300, incubated overnight with hybridization bags, HRP-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, Calif.) dilutions (1: 3000) were prepared, ECL chemiluminescence was performed in the dark for 1-10 min on X-ray films after development and fixation, discrimination was done, scanning on a gel imaging system was analyzed for absorbance values accumulated with the software of medium-Pro Plus 6.0 (America Cys, USA) and absorbance values were expressed as ratios of protein of interest (β -Santa Cruz, USA, representing the absorbance of protein of interest, Santa Cruz, Inc., Biotechnology, USA.
(6) Statistical analysis: continuous variables are expressed in mean +/-SD, multiple groups of data are subjected to variance analysis after single-factor variance analysis, homogeneity of variance test and normal distribution evaluation, pairwise comparison among the groups is tested by an LSD-T method, and when P is less than 0.05, the difference has significant meaning. All data were statistically analyzed using SPSS17.0 (SPSS corporation, chicago, usa).
Results
(1) Primary cultured BMSCs forms
BMSCs cells of primary culture are round under a mirror, are different in size, are dense in suspension cells, and begin to adhere to the wall after 4-6 hours, most BMSCs adhere to the wall after 24 hours, and cells which do not adhere to the wall are removed by changing liquid for the first time after 48 hours. After 3-4 days, the cells grow in colonies, gradually converge into slices, the morphology of the cells is changed into short spindle shapes or needle tip shapes, nucleolus can be seen, and the cells are basically paved on the bottom of the bottle for 7-10 days.
After passage, the cells are completely attached within 24h, the cell volume of the 3 rd generation is larger than that of the primary cells, most of the cells are arranged in a fusiform or fibrous shape, cytoplasm is transparent, and nucleus is obvious (as shown in figure 1, figure 2 and figure 3).
(2) Identification of surface antigens of BMSCs
The positive cell rates of BMSCs surface antigen CD90 and CD11b and CD45 are both more than 98% and less than 2% respectively through flow cytometry (as shown in figures 4, 5, 6 and 7), and the results show that the cell uniformity is good and the purity is high.
(3) Expression of phosphorylated proteins ERK and p-38 in BMSCs after induction of effective parts of Yang tonifying and five returning decoction
The ERK protein phosphorylation level at day 28 in the apr-BYHDD group is higher than that of the blank control group and the negative control group, and the ERK protein phosphorylation level at day 28 is higher than that of the 7 days and 14 days, and the difference is statistically significant (the difference is shown in the specification)P< 0.05, table 1 in fig. 8); no statistical differences were seen in the other groups. The p-38 phosphorylated protein levels in the apr-BYHDD groups at 7 days, 14 days and 28 days were higher than those in the blank control group and the negative control group (ii) ((ii))P< 0.05, FIG. 9 Table 1), wherein no significant difference was observed at 7 days, 14 days, 28 days after the induction thereof.
(4) Effect of MAPK pathway blockers on NSE expression following apr-BYHDD Induction of BMSCs
NSE mRNA expression was observed in the apr-BYHDD control group at 7 days, 14 days, and 28 days, and in the apr-BYHDD + SB203580 group, the expression was weaker at 14 days and 28 days than in the apr-BYHDD control group, and the difference was statistically significant (the difference is: (P< 0.05, FIG. 10, Table 2), the expression was lower than that of apr-BYHDD control in the apr-BYHDD + PD98059 group for 7 days, 14 days and 28 days ((B)P< 0.05, FIG. 10, Table 2). The expression of BMSCs NSE protein induced after MAPK channel blockage detected by Western-Blots is lower than that of apr-BYHHD control in 7 days, 14 days and 28 days in apr-BYHDD + SB203580 group and apr-BYHDD + PD98059 group (the expression is lower than that of apr-BYHHD control in 7 days, 14 days and 28 days: (the expression is lower than that of apr-BYHHD control in the same way in Western-BlP< 0.05, FIG. 11, Table 2) was essentially consistent with NSE mRNA expression.
(5) Effect of MAPK pathway blockers on Nestin expression following apr-BYHDD Induction of BMSCs
Nestin mRNA expression was observed in the apr-BYHHD control group at all days 7, 14 and 28, and in the apr-BYHHD + SB203580 group at all days 28, the expression was weak and poor compared with the apr-BYHHD control groupThe difference has statistical significance: (P< 0.05, FIG. 12, Table 3), was not expressed in the apr-BYHDD + PD98059 group for 7 days, 14 days, and 28 days. BMSCs Nestin protein expression induced after MAPK pathway blockage detected by Western-Blots is not expressed in the apr-BYHDD + SB203580 group and the apr-BYHDD + PD98059 group at 7 days and 14 days, and a small amount of expression is lower than that of the apr-BYHDD control at 28 days (the expression level is lower than that of the apr-BYHDD control at 28 days) (B-Blots)P< 0.05, FIG. 13 Table 3) was essentially identical to Nestin mRNA expression.
The differentiation of BMSCs into nerve cells under specific environment (induction condition) has been confirmed, and the research of the directional differentiation mechanism thereof is currently one of the hot spots of biological research. In the above experiment we first observed MAPK phosphorylated protein expression in BMSCs after apr-BYHDD induction, and found that p-p38 phosphorylation increased at 7 days after induction and peaks at 14 and 28 days. The p-ERK was increased at 28 days compared to the control group. It was suggested that apr-BYHWD had activated the MAPK pathway and thereby performed its function associated with cell proliferation and differentiation. The p38MAPK pathway was activated earlier than the MEK-ERK pathway.
Next we chose 2 different cell signaling pathway blockers PD98059 and SB203580 to block MEK-ERK and p38MAPK signaling pathways, respectively. The results suggest that NSE, 7, 14, 28 days after blocking MEK-ERK pathway, showed a decrease in gene level and protein level, whereas Nestin showed no mRNA expression, and a small amount of protein expression was detected at 28 days. NSE mRNA expression is reduced at 14 days and 28 days after the p38MAPK pathway is blocked, and the protein expression is reduced at 7 days, 14 days and 18 days. Nestin mRNA and protein were not detected expressed on days 7 and 14, and were expressed in small amounts on day 28. The MAPK pathway is shown to activate and influence the expression of NSE and Nestin when apr-BYHWD induces BMSCs. The MEK-ERK and p38MAPK signaling pathways are suggested to be involved in the apr-BYHDD neural directed differentiation process of BMSCs.
And (4) conclusion: the effective part formula of the yang-tonifying five-decoction provided by the invention can induce BMSCs to differentiate into neuron-like cells in vitro, MEK-ERK and p38MAPK signal pathways participate in the process of inducing BMSCs to differentiate into nerve cells by apr-BYHDD, and the blocking of MEK-ERK and p38MAPK signal pathways inhibits the apr-BYHDD from inducing BMSCs to differentiate into nerve cells.
Claims (4)
1. The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells is characterized in that the effective part group of the yang tonifying and five returning decoction is as follows: glycoside compounds, total alkaloids, angelica polysaccharide, safflower yellow and naringenin; the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: extracting radix astragali, radix Paeoniae Rubra and Lumbricus with ethanol under reflux, and filtering to obtain residue A; recovering ethanol from the filtrate, adjusting to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adsorbing the concentrated solution with macroporous resin column, washing with water, eluting with ethanol, collecting ethanol eluate, concentrating, and drying to obtain glycosides and naringenin effective components;
(2) separating and extracting the effective part of the safflower yellow: decocting semen Persicae and Carthami flos in water, filtering, mixing filtrates, concentrating to obtain concentrated solution with concentration of 0.5-3g medicinal materials per 1ml, adsorbing with macroporous resin column, washing with water, eluting with ethanol, collecting eluate, concentrating, and drying to obtain safflower yellow effective component;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in water, filtering to obtain filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 45-60%, stirring, standing, filtering to obtain precipitate B, volatilizing ethanol from the filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 80-85%, stirring, standing, collecting precipitate, drying to obtain effective fraction of Angelica polysaccharide, collecting ethanol precipitation solution, volatilizing ethanol, adding precipitate B for dissolving, adding water to adjust to concentrate solution of 0.5-3g medicinal material per 1ml, adsorbing with D101 macroporous resin column, washing with water, eluting with ethanol, collecting eluate, concentrating, and drying to obtain effective fraction of total alkaloids;
the types and the contents of the medicinal materials used for preparing the effective part group of the yang tonifying and five returning decoction are as follows: 23-66 g of astragalus root, 6-17 g of red peony root, 6-10g of ligusticum wallichii, 9-19 g of angelica, 9-17 g of earthworm, 6-16 g of safflower and 9-20 g of peach kernel.
2. The application of the effective part group of the yang tonifying and five returning decoction in inducing bone marrow mesenchymal stem cells to differentiate towards nerve cells is characterized in that the preparation method of the effective part group of the yang tonifying and five returning decoction comprises the following steps:
(1) separation and purification of effective parts of the glycoside compounds and naringenin: percolating radix astragali, radix Paeoniae Rubra and Lumbricus with ethanol, collecting percolate, and collecting residue A; recovering ethanol from percolate, adjusting to concentrate of 0.5-20g medicinal materials per 1ml, adding the concentrate to the top of polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, collecting 100ml eluate as a fraction, monitoring dynamic change of components of the eluted fraction by high performance liquid chromatography, mixing fractions with similar components as a component, collecting fractions of effective parts of astragaloside, paeoniflorin and naringenin, concentrating, and drying to obtain glycosides and naringenin effective parts;
(2) separating and extracting the effective parts of the safflor yellow and the naringenin: decocting semen Persicae and Carthami flos in water, filtering, mixing filtrates, concentrating to 1ml concentrated solution equivalent to 0.5-3g medicinal materials, adding the concentrated solution to the top of polyamide column, sequentially eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol, collecting 100ml eluate as a fraction, monitoring dynamic change of eluted fraction components by high performance liquid chromatography, collecting fractions of safflor yellow and naringenin, concentrating, and drying to obtain effective fractions of safflor yellow and naringenin;
(3) extracting effective parts of angelica polysaccharide and total alkaloid: decocting residue A, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in water, filtering, mixing filtrates, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 45-60%, stirring, standing, filtering to obtain precipitate B, volatilizing ethanol from filtrate, concentrating to obtain fluid extract with relative density of 1.02-1.10 at 40-60 deg.C, adding ethanol to make ethanol content reach 80-85%, stirring, standing, collecting precipitate, and drying to obtain polysaccharide effective component; taking the alcohol precipitation solution, volatilizing ethanol, adding precipitate B for dissolution, adding water to adjust to 1ml of concentrated solution equivalent to 0.5-3g of medicinal materials, adding the concentrated solution to the top of a polyamide column, eluting with pure water, 20%, 40%, 60%, 80% and 95% ethanol in sequence, taking 100ml of eluent as a fraction, monitoring dynamic change of components of the eluted fraction by a high performance liquid chromatography, combining fractions with similar components as a component, collecting fractions of effective parts of ligustrazine and ferulic acid, concentrating and drying to obtain effective parts of total alkaloids;
the types and the contents of the medicinal materials used for preparing the effective part group of the yang tonifying and five returning decoction are as follows: 23-66 g of astragalus root, 6-17 g of red peony root, 6-10g of ligusticum wallichii, 9-19 g of angelica, 9-17 g of earthworm, 6-16 g of safflower and 9-20 g of peach kernel.
3. The use of the effective fractions of BUYANGHUANWUTANG in inducing differentiation of bone marrow mesenchymal stem cells into nerve cells according to claim 1 or 2, wherein the use of the effective fractions of BUYANGHUANWUTANG in the treatment of nervous system diseases comprises the use of the effective fractions of BUYANGHUANWUTANGHUANGJIAO in preparing medicines for promoting the transformation of bone marrow mesenchymal stem cells in vitro into neuron-like cells, and the bone marrow mesenchymal stem cells are directionally differentiated into neuron-like cells.
4. The use of the effective site group of BUYANGHUANWUTANG in the medicament for treating nervous system diseases according to claim 3, wherein in the process of differentiating the mesenchymal stem cells into neuron-like cells, MEK-ERK and p38MAPK signal pathways are involved in the process of inducing BMSCs to differentiate into nerve cells from the effective site group of BUYANGHUANWUTANG, and the blockade of MEK-ERK and p38MAPK signal pathways can inhibit the combination of the effective site groups of BUYANGHUANWUTANG from inducing BMSCs to differentiate into nerve cells.
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| Title |
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| Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells;Jinghui Zheng, Jian Liang, Xin Deng;《NEURAL REGENERATION RESEARCH》;20120625;第7卷(第18期);第1370-1377页 * |
| The active principle region of Buyang Huanwu decoction induced differentiation of bone marrow-derived mesenchymal stem cells into neural-like cells: Superior effects over original formula of Buyang Huanwu decoction;Jinghui Zheng，Yi Wan, Jianhuai Chit;《NEURAL REGENERATION RESEARCH》;20120225;第7卷(第4期);第261-267页 * |
| 补阳还五汤诱导大鼠骨髓间充质干细胞向;张运克，张振强，高峰等;《2013年度中华中医药学会科学技术获奖奖项项目简介》;20131116;第61页 * |
| 骨碎补有效成分柚皮疳对人骨髓间充质;邓展生，张璇，邹冬青等;《湖南学院学报》;20051225;第7卷(第4期);第5-7页 * |
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