| BackgroundCoronary artery heart disease(CHD),as a common clinical cardiovascular disease,has become the "first killer" that threatens human life and health,and it is a disease of atherosclerosis(AS).In a large proportion,the incidence has increased year by year.Currently,it has been demonstrated that CHD is a systemic immune and chronic inflammatory disease of the coronary arteries,and a variety of immune cells participate in the entire process of stable and vulnerable AS.Recent studies have suggested that dendritic cells(DCs)are one of the major immune cells involved in the AS immune response.They play an important role in the activation of T cells and the presentation of lipid antigens in the AS.The aggregation and increase of DCs can promote Plaque progression and instability.Against this background,it is of great significance to explore the prevention and treatment of CHD by Chinese medicine from the perspective of immunology.According to the etiology,disease location and pathogenesis classification,CHD belongs to the category of traditional Chinese medicine chest pain,the clinical manifestations are rapid onset,changes in conditions,similar to Chinese medicine wind syndrome,according to many years of clinical experience,Professor Wang Xian proposed atherosclerosis The theory of " edogenous collaterals wind"has created a method which named Luoheng Prescription,activating blood and meridians,and has achieved good clinical efficacy.In previous studies,it has been confirmed that Luoheng Prescription has certain anti-inflammatory and pharmacodynamic basis.PurposeUnder the influence of large-,medium-,and small-concentration drug-containing serum of Luoheng Prescription,the changes of the characteristic molecular markers and the differences between the groups during the immune maturation of DCs before and after the interventions were observed and provided for the treatment of CHD with activating blood and meridians.Certain scientific basis to further support the theory of " edogenous collaterals wind " pathogenesis.Methods①Preparation of Luoheng Prescription drug-containing serum:Thirty experimental rats were randomly divided into groups and were dosed in accordance with kilogram body weight of human and animals.After large-,medium-,and small-concentration solutions of Luoheng Prescription granules were administered to rats 7 days after intragastric administration Blood samples were collected from abdominal aorta to prepare drug-containing serum for cell experiments;②People DCs sorting and training:selected coronary angiography(CAG)confirmed 8 cases of CHD patients,2 healthy volunteers,through the peripheral blood mononuclear cells(peripheral blood mononuclear cell,PBMC),use Immune magnetic beads method:CD 14+monocytes from human PBMCs were purified and inoculated into culture flasks.Each day,a half volume of 100ng/ml rhGM-CSF and 50ng/ml hIL-4 containing 10%fetal bovine serum 1640 was used,observe DCs cell growth in time before and after changing liquids;③ The experiments were divided into control group(DC + PBS group),DC + ox-LDL group,DC + ox-LDL + normal group serum,DC + ox-LDL + Luoheng Prescription large concentration group serum,DC + ox-LDL + Luoheng Prescription medium concentration group serum,DC+ox-LDL+ Luoheng Prescription small concentration group serum,DCs were induced with ox-LDL as an antigen.After 24 hours of culture,the morphology of DCs in each group was observed by an inverted microscope.The ultrastructure of DCs was observed by scanning electron microscopy,and the activity of cells was determined by trypan blue staining.The MTT method was used to observe the network of different Luoheng Prescription concentrations.The influence of the intervention of Luoheng Prescription on the proliferation ability of DCs was tested by flow cytometry,and the expression of CD1a、CD80、CD83 and CD86 on the surface of DCs after the intervention of Luoheng Prescription was detected.Results①During the process of preparation of serum containing Luoheng Prescription,no rats died and the collection of drug-containing serum was completed;②The purity of the separation by immunomagnetic beads method is high,and each group is more than 90%.Microscopically,most of the recently isolated and purified CD 14+mononuclear cells were in the adherent state with smaller volumes and higher purity.The nucleus was round or elliptical.After 24 hours of cultivation,the cells were scattered and evenly distributed,and they gradually separated and suspended.On the third day of culture,the cells were semi-affixed to a semi-suspended state.The cell volume gradually increased and the number increased.Some cells aggregated to form cell clusters.On the sixth day of culture,most cells were suspended and a few cells adhered.Some of the cells had looms around the burrs,which were unevenly distributed and the nucleus was deviated.DCs had initially formed③After being divided into groups,the number of DCs without added Luoheng Prescription was significantly increased,and a large number of cluster-like burrs grew around the cell membrane.The number of cells in the Luoheng Prescription group decreased,and the differentiation maturity was lower than that of the ox-LDL group alone.The ultrastructure of DCs was observed under scanning electron microscope.The cell suspension growth was observed under electron microscope at 1000 X.The structure was intact and the nucleus was shifted.Except the PBS group,the remaining five groups showed rough cell surface,irregularities,and obvious dendritic structure on the cell membrane.The dendritic structure was reduced in the intervention group compared with the model group and the normal serogroup,and it was the most significant in the small concentration group.3000 X electron microscopy shows that the model group and the normal serum group similarity is larger,the cytoplasm is longer,there are many depressions,a large number of burr-like protrusions on the cell surface,slender and dense.In the large concentration group,the cell surface was rough,there were a large number of lamellar folds,the surface burrs on the cell membrane were short and slightly blunt,showing a peripheral distribution,the cell surface folds in the medium and small concentration groups were reduced,and the dendritic structure was less;The blue staining results showed that the DCs extracted from peripheral blood of 8 patients with coronary heart disease were well differentiated by ox-LDL and the survival rate was greater than 85%;The MTT assay showed no significant difference in cell viability,and there were continuous grouping experimental conditions.As the serum drug concentration continues to increase,OD values slowly decline,and do not exclude the existence of potential cytotoxicity.④The expression of CD1a、CD80、CD83and CD86 on the surfaces of DCs induced by ox-LDL significantly increased compared with the control group(P<0.01);the specific molecules of the control serum group compared with the model group.There was no significant change in the expression of markers(CD1a、CD80、CD83、CD86),and the difference was not statistically significant(P>0.05),which was in line with the conditions for continuous group intervention with Luoheng Prescription.After intervention of Luoheng Prescription large,medium and small concentrations of serum,the expression of CD1a、CD80、CD83 and CD86 of DCs was down-regulated,and the difference was statistically significant(P<0.05 or P<0.01).The most significant effect of serum down-regulation was observed in serum of medium concentration group and serum of large concentration group again.There was significant difference between groups(P<0.01).ConclusionLuoheng Prescription can inhibit the activation and maturation of DCs and the expression of characteristic molecular markers(CD1a、CD80、CD83、CD86),providing a scientific basis for the clinical application of the "edogenous collaterals wind" pathogenesis. |
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