Study On The Characteristics Of Oxidative Stress Factors In Dyslipidemia, Phlegm-turbidity Inhibition Syndrome And Spleen-kidney Yang Deficiency Syndrom

With the rapid development of social economy,the change of people’s diet structure and the gradual reduction of physical activity,the number of patients with dyslipidemia has gradually increased.The disorder usually refers to elevated levels of cholesterol,triglycerides,and abnormalities in LDL and HDL cholesterol.Due to its damaging effect on vascular endothelial cell(VEC),the disease is not only the pathological basis of atherosclerosis,but also an important risk factor for cardiovascular and cerebrovascular diseases.Clinically,our research group found that in the disease state of dyslipidemia,there are more patients with phlegm turbidity syndrome and spleen and kidney yang deficiency,so the research group has been working on phlegm turbidity syndrome and spleen and kidney yang deficiency in dyslipidemia In-depth research on syndromes and syndromes,the results of pre-menstrual studies found that differences in syndromes can be obtained from the perspective of blood and oxidative stress-related indicators.Among them,the oxidative stress process can reflect the syndrome characteristics of dyslipidemia phlegm turbidity syndrome and spleen-kidney yang deficiency syndrome.In the state of dyslipidemia,there is a close relationship between the process of dyslipidemia and vascular endothelial dysfunction.It is feasible to study the characteristics of different syndromes of dyslipidemia from the perspective of endothelial injury.Therefore,this study will provide a basis for the treatment of dyslipidemia with traditional Chinese medicine through the combined analysis of clinical and animal model experimental studies.Objective:1 Clinical research:Aims to study the differences and characteristics of serum nuclear factorκB(NF-κB)and endothelin-1(ET-1)levels between patients with dyslipidemia and phlegmturbidity suppression syndrome and spleen-kidney yang deficiency by clinical crosssectional study.To study the correlation between the degree of syndromes and NF-κB and ET-1 levels in patients with dyslipidemia and phlegm-turbidity suppression syndrome and spleen-kidney yang deficiency.By combining TCM syndromes with modern medical indicators,this study sought the correlation between physical and chemical indicators and the degree of syndrome,analyzed and elucidated its possible mechanism,and explored new ideas for the prevention and treatment of primary dyslipidemia by TCM.2 Animal experiment:In order to observe the characteristics and similarities of serum TC,TG,LDL-C and HDL-C in ApoE-/-mice with dyslipidemia,phlegm-turbidity suppression syndrome and spleen-kidney yang deficiency,the animal models of dyslipidemia,phlegmturbidity suppression syndrome and spleen-kidney yang deficiency were constructed by multi-factor compound modeling method.The characteristics and similarities of serum oxidative stress factor(NF-κB,NO)in ApoE-/-mice with dyslipidemia and phlegm-turbidity suppression syndrome and spleen-kidney yang deficiency were observed.The characteristics and similarities of NF-κB staining in aorta of ApoE-/-mice with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency were observed.Through this experiment,the changes of NF-κB signal pathway related factor(NF-κB)inflammatory expression level in vascular endothelial NF-κB aortic immunohistochemical staining were further analyzed.This study investigated the expression characteristics of oxidative stress factors in animals with dyslipidemia and phlegm-turbid suppression syndrome and spleen-kidney yang deficiency,which can provide experimental basis for clinical application of traditional Chinese medicine in the treatment of dyslipidemia and prevention of cardiovascular and cerebrovascular diseases.Method:1 Clinical research part:Inpatients of the First Clinical Medical College of Beijing University of Chinese Medicine(Dongzhimen Hospital)from January 2018 to July 2018 were selected.A total of 78 patients who met the inclusion criteria of this study were collected according to the corresponding inclusion and exclusion criteria,and informed consent was signed.In this way,the general information of patients could be collected,and the traditional Chinese medicine syndrome observation form of patients with dyslipidemia could be filled out.39 cases of sputum stasis syndrome and spleen-kidney yang deficiency were selected.Venous blood samples were collected from patients with fasting for more than 8 hours(in the morning of the day of admission or the next morning).After the collected blood was tested for four items of lipid(TC,TG,LDL-C,HDL-C),serum was separated from the remaining venous blood in time and stored in EP tubes.The samples were frozen at-80℃ for the determination of serum ET-1 and NF-κB.2 Animal experiment:A total of 10 6-week-old male ApoE-/-mice were used to establish the animal model of disease syndrome(based on the previous study of the research group)by multi-factor compound modeling method.There were 5 mice in the sputum stasis syndrome group and 5 mice in the spleen-kidney yang deficiency group.Another 5 6-week-old male C57BL/6J mice of the same lineage were selected as normal control group.Normal control group mice were given ordinary diet every day for 4 weeks.ApoE-/-mice in dyslipidemia sputum stasis group were given high-fat diet daily for 4 weeks.For ApoE-/-mice of spleenkidney yang deficiency group with dyslipidemia,0.1%propyl thiouracil was given at the dose of 10 mg/(kg·d)from week 1 to week 4,and high-fat diet was continuously given for weeks 4-6.The above methods were used to construct disease-syndrome animal models to complete the construction of disease-syndrome animal models.Next,the syndrome-related manifestations of dyslipidemia phlegm-turbidity suppression syndrome and spleen-kidney yang deficiency-syndrome animal model were observed.The serum levels of TC,TG,LDL and HDL in each group were detected by oxidase method.The differences and characteristics of serum lipid in ApoE-/-mice with different syndromes of dyslipidemia were observed.The endothelial oxidative stress levels of serum NO and NF-κB were measured by ELISA.The characteristics of serum oxidative stress factors in ApoE-/-mice with different syndromes of dyslipidemia were observed in this experiment.The thoracic cavity of mice was opened,the tissue was dissected,and the aorta of mice with dyslipidemia syndrome was taken and fixed in freshly prepared 4%paraformaldehyde solution.The aorta was stained with NF-κB aortic immunohistochemistry.Results:1 Clinical research1.1 Comparison of demographic data between two groups:A total of 78 patients with dyslipidemia were included,including 26 males(33.33%)and 52 females(66.67%),with an average age of 58.7±12.26 years(rangeing 27-79 years),the minimum age was 27 years,and the maximum age was 79 years.There were 39 cases of sputum stasis syndrome and 39 cases of spleen-kidney yang deficiency.By comparing the demographic data of the patients in terms of gender,age,BMI,systolic blood pressure,diastolic blood pressure,heart rate,respiration and body temperature,there was no statistical difference in baseline demographic data between the two groups(P>0.05).1.2 Comparison of serum ET-1 and NF-κB levels between two groups of dyslipidemia patients:The serum ET-1 concentration in patients with sputum stasis syndrome was higher than that in patients with spleen-kidney yang deficiency,and the difference was statistically significant(P<0.05).The serum NF-κB concentration in patients with sputum stasis syndrome was higher than that in patients with spleen-kidney yang deficiency,and the difference was statistically significant(P<0.05).1.3 Correlation analysis of serum ET-1.NF-κB concentrations and TCM syndrome scores in patients with spleen-kidney yang deficiency of dyslipidemia:There was a positive correlation between serum ET-1 concentration and TCM syndrome score,and the correlation was not statistically significant(r=0.064,P>0.05).Serum NF-κB level was positively correlated with TCM syndrome score,and the correlation was statistically significant(r=0.340,P<0.05).1.4 Correlation analysis of serum ET-1,NF-κB concentrations and TCM syndrome scores in patients with dyslipidemia and phlegm-turbidity repressing syndrome:There was a positive correlation between serum ET-1 concentration and TCM syndrome score,and the correlation was not statistically significant(r=0.176,P>0.05).Serum NF-κB level was positively correlated with TCM syndrome score,and the correlation was statistically significant(r=0.682,P<0.05).2 Animal experiment2.1 The characteristic manifestations of the syndrome in the animal model of dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency were as follows:1)Sputum stasis syndrome:They had glossy coats,fat,sticky stools and a tendency to lie.2)spleenkidney yang deficiency:Their for color was not flourish,with bowed back curled,like clustering,with loose and soft stool,prone to lying.These results indicated that the quality of the animal models of dyslipidemia sputum stasis syndrome and dyslipidemia spleenkidney yang deficiency established in this study was satisfying.2.2 Comparison of blood lipid levels in each group:According to the analysis of experimental data,compared with the normal control group,the serum TC,TG and LDL-C levels of ApoE/-mice with dyslipidemia-syndrome were increased in the two groups.The serum levels of TC,TG and LDL-C in the spleen-kidney yang deficiency group were significantly higher than those in the normal control group.The levels were in the following order:spleen-kidney yang deficiency group,sputum stasis syndrome group,normal control group,and the difference was statistically significant(P<0.05).In terms of HDL-C index,the level of sputum stasis syndrome group was higher than that of the other two groups,but there was no statistical difference among the three groups(P>0.05).2.3Comparison of serum levels of NO in each group:In terms of NO concentration level,The serum levels of NO in the normal control group were higher than those in the two dyslipidemia-syndrome model groups.The level of normal control group,spleen-kidney yang deficiency group,sputum stasis syndrome group,and the difference was statistically significant(P<0.05).2.4 Comparison of serum NF-κB levels in each group:In terms of serum NF-κB concentration in each group,the levels of serum NF-κB in ApoE-/-mouse model of dyslipidemia in two groups were higher than those in normal control group.The level of sputum stasis syndrome group,spleen-kidney yang deficiency group,normal control group,and the difference was statistically significant（P<0.05).2.5 The results of NF-κB immunostaining of aorta of ApoE-/-mice with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency were as follows:A large number of brown granular NF-κB p65 positive cells were found in the epithelial cells of the aortic mucosa of ApoE-/mice with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency constructed by multi-element compound modeling method.The nuclear NFκB p65 staining of most inflammatory cells infiltrated in the mucosal layer was positive.A large number of inflammatory cells infiltrated in the submucosa were also NF-κB p65 positive cells.The results of NF-κB p65 positive cells in aortic endothelium of ApoE-/-mice with dyslipidemia showed the following contents:The number of NF-κB p65 positive cells in ApoE-/-mice with dyslipidemia and sputum stasis syndrome was significantly higher and their color was darker.The expression level of NF-κB in ApoE-/-mice with sputum stasis syndrome of dyslipidemia was higher than that in ApoE-/-mice with spleen-kidney yang deficiency of dyslipidemia.Conclusions:1 The serum levels of ET-1 and NF-κB in patients with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency can reflect the characteristics of their respective syndromes to a certain extent.The level of NF-κB can reflect the severity of patients’ syndrome to a certain extent.The above characteristics can provide the basis for standardization and objective syndrome differentiation.2 Based on the previous research results of the research group,the animal models of dyslipidemia sputum stasis syndrome and spleen-kidney yang deficiency are established in this study,and the model quality is satisfying.3 Serum levels of NO and NF-κB in mice with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency can reflect the characteristics of each syndrome.4 The results of immunohistochemical staining of aortic NF-κB in ApoE-/-mice with dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency show that there are differences in oxidative stress levels in different syndromes of dyslipidemia.Therefore.it can be speculated that the differences in the above syndromic characteristics may be related to the different activation levels of NF-κB signaling pathway.5 he above studies show that the syndrome characteristics of dyslipidemia and sputum stasis syndrome and spleen-kidney yang deficiency are consistent with their respective etiology and pathogenesis analysis.This provides an objective basis for the material basic research of different syndromes and enriches the scientific connotation of TCM syndrome differentiation and treatment of this disease.