| Objective:Decoction is the most common preparation form of traditional Chinese medicine,which is usually made by boiling with water as the solvent.There are not only a large number of water-soluble components,but also a complex colloidal dispersion system,which contains a large number of nanoparticles.Nowadays,nanodrugs have been utilized to improve solubility of insoluble drug,delivery of unstable drug and targeting tumor.So it is attractive to find out whether the nanoparticles in traditional Chinese medicine decoction have drug loading capacity and biological activity.First of all,we need to make clear the physical and chemical properties of Chinese medicine nanoparticles,so as to determine the possibility of drug loading capability of nanoparticles;secondly,we need to establish a suitable and sensitive animal model to evaluate the biological activity of Chinese medicine nanoparticles.In this study,Siwu Decoction,a traditional blood tonic,was selected as the research object.Siwu Decoction is composed of four kinds of traditional Chinese medicine,i.e.Rehmannia glutinosa,Angelica sinensis,Paeonia lactiflora and Ligusticum chuanxiong.It has the effect of nourishing the blood and regulating blood circulation,and can be used to treat various diseases of deficiency of blood and asthenia syndrome.It is mainly used in the treatment of anemia,dysmenorrhea,angina pectoris,diabetes and other diseases in clinic.Modern pharmacological research shows that Siwu Decoction has many biological activities on blood system,which provides a positive reference for the biological activity of Siwu Decoction nanoparticles.Firstly,the extraction and separation method of nanoparticles in Siwu Decoction was established.From the perspective of nano dispersion system and drug aggregation,Siwu Decoction nanoparticles were studied to explore the physical characteristics and chemical composition of nanoparticles.The size,morphology and stability of Siwu Decoction nanoparticles were characterized.Meanwhile,the content of major biological macromolecule such as protein,polysaccharide and DNA in Siwu Decoction nanoparticles was determined.In order to reduce the difficulty of drug preparation and ensure sufficient dosage,zebrafish(Danio rerio)was selected as the model animal.In order to clarify the pharmacological activities of Siwu Decoction and its nanoparticles,the radiation damage model of adult zebrafish induced by ionizing radiation was used to evaluate the effects of Siwu Decoction and its nanoparticles on the blood system of adult zebrafish.And the model of zebrafish embryo hemolytic anemia induced by phenyl-hydrazine was used to evaluate the effects of Siwu Decoction and its nanoparticles on the blood system of zebrafish embryo.Methods:1.Extraction,separation and characterization of physical and chemical properties of Siwu Decoction nanoparticles:the nanoparticles of Siwu Decoction were separated by a series of centrifugations.The size,morphology and stability of Siwu Decoction nanoparticles were characterized by transmission electron microscopy and dynamic light scattering.The protein content of Siwu Decoction and its nanoparticles was determined by Bradford method,the polysaccharide content was determined by phenol-sulfuric acid method,the DNA was extracted by kit,and the DNA content was determined by UV spectrophotometry method.2.Establish blood system injury model by radiation-induced in adult zebrafish:Adult zebrafish(3-5 months old,0.14-0.20 g in weight,male)was exposed to 60Co-? ray at a dose of 20 Gy and a dose rate of 97.33 cGy/min.Adult zebrafish was dissected respectively on the 3rd,7th and 14th day after post irradiation.And the kidney marrow cells and peripheral blood cells were collected.The number and percentage of erythrocytes,myelomonocytes,precursors and lymphocytes in the kidney marrow and peripheral blood cells of zebrafish were analyzed by flow cytometry.3.Study on the dosage of Siwu Decoction in adult zebrafish:The condition of adult zebrafish is the same as above.After normal zebrafish and irradiated zebrafish was been treated with different concentrations of Siwu-Decoction,the survival rate was observed and the minimum lethal dose of the drug was determined.After zebrafish was treated with Siwu Decoction of different concentrations for 5 days,the animals were dissected and the kidney marrow cells and peripheral blood cells were collected.The changes of the number and percentage of erythrocytes,myelomonocytes,precursors and lymphocytes in the kidney marrow and peripheral blood cells of zebrafish were analyzed by flow cytometry.So as to determine the safe concentration range of Siwu Decoction.4.The protective effect of Siwu Decoction and its nanoparticles on injury induced by radiation in adult zebrafish:Refer to the above-mentioned conditions of zebrafish mode and the safe concentration range of Siwu Decoction,zebrafish was treated 7 d with appropriate concentration of Siwu Decoction and its nanoparticles.And kidney marrow cells and peripheral blood cells were collected.The changes of the number and percentage of erythrocytes,myelomonocytes,precursors and lymphocytes in the kidney marrow and peripheral blood cells of zebrafish were analyzed by flow cytometry.5.Establish the hemolytic anemia model of zebrafish embryos induced by phenyl-hydrazine:52 hpf zebrafish embryos were treated with phenyl-hydrazine of different concentrations in the range of 0.005-0.1?g/ml for 24 hours,and then ODA staining was carried out to determine the appropriate concentration of phenyl-hydrazine.6.Study on the dosage of Siwu Decoction in zebrafish embryos:zebrafish embryos were treated with different concentrations of Siwu Decoction,then ODA staining was carried out to determine the appropriate dosage.7.The effect of Siwu Decoction and its nanoparticles on phenyl-hydrazine(PHZ)-induced hemolytic anemia in zebrafish embryos:According to the above-mentioned conditions of zebrafish embryos model and the safe dose range of Siwu Decoction,zebrafish embryos were treated with appropriate concentration of Siwu Decoction and its nanoparticles and then ODA staining was carried out.Microscopes were used to take photos and record videos,and the results were evaluated by observing total blood flow,heart rate,pericardial edema area,and the number of gatal+cells and related gene expression.8.Transcriptome analysis:Siwu Decoction and its nanoparticles were given to zebrafish embryo hemolytic anemia model induced by phenylhydrazine and differential expression gene analysis,go enrichment analysis and KEGG enrichment analysis were carried out.Results:1.The results of extraction,separation and physical characterization of Siwu Decoction nanoparticles show that the extraction yield of NP20000 and NP100000 nanoparticles was 0.05%and 0.03%respectively.NP20000 and NP100000 nanoparticles were circular or irregular in shape under TEM.And the particle size of nanoparticles was about 50-500 nm.The average particle size measured by DSL was 423.0 nm and 298.3 nm respectively,and Pd1 was 0.23 and 0.30 respectively,which were moderate dispersion.The zeta potential of NP20000 and NP 100000 was-35.8 MV and-30.3 MV respectively,which indicated that the samples were relatively stable.Moreover,the particle size of NP20000 at 1-8 d did not change significantly.Besides,the protein content of Siwu Decoction nanoparticles NP20000 and NP100000 was 11.20%and 13.53%;the polysaccharide content was 36.10%and 56.70%;the DNA content was 1.19 ‰ and 1.07 ‰ respectively.2.The results of establishment of model of radiation-induced blood system injury in adult zebrafish showed that on the 7th day after post irradiation,kidney marrow cells,lymphocytes,precursors,myelomonocytes and erythrocytes decreased by 52%,79%,49%,46%and 33%respectively,which was significantly different from that of the control group;on the 14th day,compared with the 7th day,lymphocytes and myelomonocytes increased by more than 70%,and precursors and erythrocytes also increased slightly;on the 30th day,all kinds of cells exceeded the number before irradiation,and the number of precursors and myelomonocytes increased up to 178%and 112%before post irradiation,and lymphocytes and erythrocytes increased 51%and 16%respectively.At 7-30 days after post irradiation,the percentage of cells in kidney marrow was more complex.The percentage of erythrocytes and myelomonocytes increased early and then decreased.The percentage of lymphocytes decreased first and then increased.The percentage of precursors did not change during 14 days,but only increased at 30th day.The number of peripheral blood cells(mainly red blood cells)decreased by 26%on the 7th day after post irradiation,then increased to normal level on the 14th day,and exceeded the number before post irradiation on the 30th day.3.The results of adult zebrafish dosages showed that Siwu decoction at the diluted concentrations of SW100,SW200 and SW600 could cause the death of adult zebrafish,and SW600 was the minimum lethal dose.The concentrations range of SW1000-SW20000 did not affect the survival of zebrafish and did not cause obvious toxicity to various cells of kidney marrow and peripheral blood cells,so it was an appropriate concentration for drug administration.4.The protective effect of Siwu Decoction and its nanoparticles on radiation injury of zebrafish results showed that the number of whole kidney marrow cells and four kinds of cells in the model group decreased significantly.Compared with that of model group,the total number of kidney marrow cells,myelomonocytes,precursors,lymphocytes and erythrocytes increased by 57%(p=0.001),125%p=7.35×10-8),81%(p=0.001),59%(p=0.03)and 35%(p=0.019)respectively.In NP2000 group,the total number of kidney marrow cells,myelomonocytes,precursors,lymphocytes and erythrocytes increased by 16%,46%,11%,11%and 7%,respectively.The number of myelomonocytes had significant difference(p=0.011).5.Establishment the hemolytic anemia model of zebrafish embryos induced by PHZ.The results showed that the concentration of PHZ in the range of 0.02 g/mL to 2.5 g/mL could reduce the staining area of ODA,which represented a decrease in the number of red blood cells.By comparison,0.02 g/mL was considered as the optimum molding concentration.6.Determination of drug dose for zebrafish embryos.The results showed that SW100 concentration could lead to the death of zebrafish embryos and no embryo death,malformation or other abnormalities were found in zebrafish embryos treated with SW500-SW10000 concentrations range.SW500-SW10000 is the safe concentrations range of Siwu Decoction for zebrafish embryos.7.The effect of Siwu Decoction and its nanoparticles on PHZ-induced hemolytic anemia in zebrafish embryos.The results showed that SW1000 and NP250 treatment significantly increased blood flow in PHZ-induced hemolytic anemia in zebrafish embryos,and SW500,SW1000 and NP250 treatment significantly increased the number of gatala+cells and the number of gatala+cells flowing through the tail vessels.Treatment with NP250,NP1000 and NP5000 significantly reduced the area of pericardial edema in zebrafish embryos.The effect of Siwu Decoction and its nanoparticles on the gene expression of alas2,epo,pu.1,mpegl and rag2 in zebrafish embryo hemolytic anemia model induced by PHZ.Compared with the model group,the expression of alas2 in SW500 group and NP250 group was significantly up regulated(P<0.001);compared with the model group,the expression of epo in SW500 group and NP250 group was significantly down regulated(P<0.01);compared with the model group,the expression ofpu.1 in NP250 group was significantly down regulated(P<0.01);compared with the model group,the expression of rag2 in SW500 group and NP500 group was significantly down regulated(P<0.001).There was no significant difference in the expression of mpeg1 between the SW500 and NP500 groups compared with the model group(P>0.05).8.Compared with model group,there were 49 differentially expressed genes in SW500 group,among which 23 genes were up regulated and 26 genes were down regulated.Compared with model group,NP250 group had 30 differentially expressed genes,among which 15 genes were up regulated and 15 down regulated.Through go enrichment analysis,the differentially expressed genes of SW500 and model groups and np250 and model groups were enriched into four identical pathways:carbohydrate metabolic progress,heme binding,tetrapyrrole binding and cofactor binding.There were significant differences in the concentration of differentially expressed genes in 5 pathways between SW500 group and model group(P<0.05).They are spliceosome,protein processing in endoplasmic reticulum,MAPK signaling pathway,ribosome and endocytosis.In NP250 group and model group,there were three different pathways(P<0.05),which were spliceosome,protein processing in endoplasmic reticulum and endocytosis.Conclusion:1.Siwu Decoction nanoparticles with particle size distribution of 100-1000 nm can be obtained by differential centrifugation.The physical properties indicate that Siwu Decotion nanoparticles can form a relatively stable colloidal dispersion system.The particle contains large amounts of proteins,polysaccharides and very little DNA.The macromolecules as carrier for nanoparticles formation provides a probability.2.After post irradiation to zebrafish adults with 60Co-? ionizing radiation at 20 Gy dose and 97.33 cGy/min dose rate,it can cause reversible damage to zebrafish blood system,which can be self-repaired within 30 days.3.Siwu Decoction SW5000 and Siwu Decoction nanoparticles NP2000 can promote the regeneration of zebrafish hematopoietic function damage of kidney marrow caused by radiation,and Siwu Decoction nanoparticles can protect-peripheral blood cells from radiation.4.The administration of 0.02 mg/mL of PHZ in zebrafish embryos resulted in a moderate hemolytic anemia model in zebrafish embryos.5.Siwu decoction and its nanoparticles have a protective effect on blood cells in the model of hemolytic anemia of zebrafish embryos induced by PHZ,while reducing its cardiac toxicity.6.After transcriptome sequencing analysis of control group,model group,SW500 group and NP250 group,it was found that the results of go enrichment analysis showed that Siwu Decoction and its nanoparticles involved in the biological process were closely related to heme synthesis,degradation and protein binding.The enrichment analysis of KEGG signaling pathway showed that Siwu Decoction and its nanoparticles mainly participated in the following signaling pathways:spliceosome,protein processing in endoplasmic reticulum,endocytosis,etc.Overall,the experiments shows that zebrafish could be a suitable blood system model for traditional Chinese medicine investigation;the water-soluble components of Siwu decoction and its nanoparticles contribute to the blood system efficacy of Siwu Decoction. |
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