Study On The Effect Of Luofengning No.1 Prescription On The Function Of Dendritic Cells And Its Mechanism

BackgroundCoronary atherosclerotic heart disease(CHD)is a disease that seriously endangers human health,and its basic pathological change is atherosclerosis(AS).With the continuous advancement of modern medicine,research on the mechanism of AS is very extensive.The occurrence and development of AS involves inflammation,immunity,blood coagulation,neuro-endocrine regulation and other links.How to prevent and treat AS in the early stage is an important proposition.Out of holistic view of Traditional Chinese Medicine(TCM),TCM's understanding of the pathogenesis of coronary heart disease has its unique advantages in preventing and treating AS.According to the evolution of clinical diseases,Professor Wang Xian proposed the theory of internal pathogenesis of collateral wind,and created Luofengning No.1 prescription with the effects of dispelling wind,dredging collaterals,activating blood and relieving pain.Clinical trials have confirmed its curative effect on coronary heart disease.Animal experiments and cell experiments proved that Luofengning No.1 prescription has the effects of anti-inflammatory,regulating blood lipids and improving vascular endothelial function.Dendritic cells(DCs)are powerful antigen-presenting cells that play an important role in the pathological process of AS.In the process of AS,DCs are immune to maturation under the action of antigens,activate T cells through antigen presentation,promote T cell proliferation and differentiation,and then promote the production and recruitment of macrophages,release inflammatory factors,promote foam cell production,and accelerate AS process.In this experiment,DC was selected as the research object,and Luofengning No.1 prescription was used to intervene in the maturation process of DC,to explore its influence on DC immune function and JAK/STAT/SOCS signaling pathway.This experiment is to clarify the molecular mechanism of Luofengning No.1 prescription against AS,and provide new ideas and methods for the research of TCM preventing and treating cardiovascular diseases.PurposeOn the one hand,this Meta-analysis analyzes the published clinical research and explores the effectiveness of treating coronary heart disease with the wind theory.On the other hand,cell experiment was used to explore the mechanism of Luofengning No.1 prescription in preventing and treating AS.In this experiment,DC was selected as the research object and the important immune antigen ox-LDL in AS was used to promote its maturation,to simulate the activated state of DC in hyperlipidemia of human beings.Intervene the maturation process of DC with Luofengning No.1 prescription,and explore its influence on the immune function of DC and JAK/STAT/SOCS signaling pathway.Then compare the results with JAK inhibitor group,to explore the possible mechanism of the effects of Luofengning No.1 prescription on DC immune function.Method1.To explore the effectiveness of treating coronary heart disease with wind theory on clinical research literaturesBy searching the Chinese and English databases,the clinical research literatures on the treatment of coronary heart disease with wind theory were selected for meta-analysis.2.Preparation of rat serum containing Luofengning No.1 prescriptionThe dosage was calculated according to the kilogram body weight of humans and rats,and rats were gavaged with Luofengning No.1 prescription or normal saline.After 7 days,blood was taken from the abdominal aorta and the serum was collected for further use.3.Preparation and cultivation of DCCollect peripheral blood of healthy volunteers and use density gradient centrifugation and immunomagnetic bead sorting to obtain DC precLrsors—CD14+monocytes.The precursor cells differentiate into immature DC under the combined effect of GM-CSF and IL-4.4.DC maturation and identificationThe CCK-8 method was used to screen the concentration range and action time of ox-LDL that have a small effect on DC cell viability.Flow cytometry was used to detect the expression of DC surface marker molecules and explore the best conditions for ox-LDL to promote DC maturation.It can also be used to identify the maturity of DC.5.The effect of Luofengning No.1 prescription on DC immune functionThe CCK-8 method was used to explore the suitable conditions of JAK1/2 inhibitor ruxolitinib and rat serum with DC.The DC were divided into 6 groups:blank group,model group,ruxotinib group and Luofengning high,middle and low concentration groups.After group intervention,DCs were co-cultivated with T cells,and the Elisa method was used to detect the levels of various inflammatory cytokines in the supernatant,and to explore the effect of Luofengning No.1 prescription on the ability of DCs to activate T cells.Western Blot method was used to detect the expression of LOX-1 and CD36 protein in DC,and RT-PCR method was used to detect the expression of LOX-1 and CD36 mRNA in DC to evaluate the effect of Luofengning No.1 prescription on DC response to ox-LDL.6.The influence of Luofengning No.1 prescription on the JAK/STAT/SOCS signaling pathway in DCThe experimental groups are the same as above.To explore the influence of Luofengning No.1 Prescription on JAK/STAT/SOCS signaling pathway,the expression of related proteins in the JAK/STAT/SOCS signaling pathway was detected by Western Blot,and the expression of related mRNAs in the JAK/STAT/SOCS signaling pathway was detected by RT-PCR.Compare the results of Luofengning No.1 prescription group and the results of JAK inhibitor group,and explore the possible mechanism of Luofengning No.1 prescription affecting DC immune function.Result1.To explore the effectiveness of treating coronary heart disease with wind theory on clinical research literaturesThe literature meta-analysis results indicate that the total effective rate of the treatment of coronary heart disease(or the effective rate of angina pectoris)with wind theory is better than that of the control group,and it has improved electrocardiogram(ECG)efficacy and TCM syndrome efficacy,and reduced blood lipid level and hs-CRP,suggesting that treating coronary heart disease with wind theory can achieve a certain clinical effect.2.Concentration screening of ox-LDL,ruxolitinib,and rat serumFrom the results of CCK-8 and the detection results of DC surface flow cytometry molecules,100?g/mL ox-LDL intervention for 24h was selected as the modeling condition.The concentration of ruxolitinib on DC was 0.1 ?mol/L,and the concentration of rat serum on DC was 20%.3.Identification of DCThe results of flow cytometry showed that,compared with the blank group,the expression of CD1a,CD80,CD86 and HLA-DR on the surface of DC increased after ox-LDL acted on DC,suggesting that ox-LDL can promote DC immune maturation.4.The effect of Luofengning No.1 prescription on the function of DC activating T cellsElisa method was used to detect the cell supernatant of each group of DCs and T cells after 48 hours of co-cultivation.The results showed that compared with the blank group,the DCs of ox-LDL group can significantly activate T cells and promote the cytokines IL-4,IFN-?,IL-6 and IL-17A secretion.Compared with the model group,the levels of the aforementioned cytokines in the cell supernatants in ruxolitinib and Luofengning No.1 prescription groups were reduced in varying degrees,suggesting that the Luofengning No.1 prescription and ruxolitinib can reduce the function of DCs activating T cells.5.The effect of Luofengning No.1 prescription on the scavenger receptors of DCWestern blot and RT-PCR results show that compared with the blank group,ox-LDL can significantly up-regulate the protein expression and gene expression of LOX-1 and CD36;compared with the model group,Luofengning No.1 prescription group and ruxolitinib group can down-regulate the protein expression and gene expression of LOX-1 and CD36,suggesting that both Luofengning No.1 and ruxolitinib can attenuate the response of DC to ox-LDL.6.The influence of Luofengning No.1 prescription on the JAK/STAT/SOCS signaling pathway in DCWestern blot and RT-PCR results showed that compared with the blank group,ox-LDL can significantly up-regulate the protein and gene expression of JAK1/2 and STAT3,promote the expression of p-STAT3,and down-regulate the protein and gene expression of SOCS1:compared with the model group,Luofengning No.1 prescription,high concentration group,medium concentration group and ruxolitinib group can down-regulate JAK1/2 and STAT3 protein and gene expression,reduce STAT3 phosphorylation,and up-regulate SOCS1 protein and gene expression.It suggests that both Luofengning No.1 and Ruxotinib can inhibit the JAK/STAT signal pathway and activate the negative feedback regulation of SOCS1.Conclusion1.A meta-analysis of relevant literature shows that under the same condition that the basic western medicine treatment of coronary heart disease is the same,the treatment of coronary heart disease with wind theory can achieve a better clinical effect,and it can improve the total effective rate or the effective rate of angina pectoris.Also,it has improved ECG efficacy and TCM syndrome efficacy,and reduced blood lipid level and hs-CRP,suggesting that treating coronary heart disease with wind theory can achieve a certain clinical effect.2.Ox-LDL can promote the immune maturation of DC.Luofengning No.1 prescription can weaken the ability of DC activating T cells,reduce the secretion of inflammatory factors,weaken the response of DC to ox-LDL,and then inhibit the immune function of DC.3.Ox-LDL can activate the JAK/STAT signal pathway in DC and inhibit the negative feedback regulation of SOCS1,while Luofengning No.1 prescription can inhibit the JAK/STAT signaling pathway and activate the negative feedback regulation of SOCS.The effect of JAK inhibitor on DC is similar to that of Luofengning No.1 prescription,which suggests that Luofengning No.1 prescription affect the function of DC activating T cells and the response to ox-LDL by inhibiting JAK/STAT signaling pathway and activating the negative feedback of SOCS1.