| Objective: Exploring the curative effect evaluation and PD-1 expression in peripheral blood in atherosclerosis subjects treated by Shenxiaheji through clinical research;Exploring the effects of Shenxiaheji and Brazilin on the mRNA levels and protein expressions of PD-1,TLR-4,NF-?B and MMP-9 in THP-1 cell cultured in vitro;Based on the results of cell experiments,exploring the effects of Shenxiaheji and Brazilin on the formation of atherosclerosis plaque and the expression of CD4+T,CD8+T,macrophages and PD-1 in Apo E mice through animal experiments.Methods: 1.According to the inclusion and exclusion criteria,40 patients with AS who met the requirements were selected and randomly divided into the control group and the test group.Simvastatin group and Shenxiaheji group were treated for 4 weeks,respectively.Observing the PD-1 expression in peripheral blood,blood fat,hypersensitive c-reactive protein and carotid intima-media thickness,plaques area and TCM syndrome integral and clinical effect before and after treatment in the two groups.2.Cell experiment: The human mononuclear cell line THP-1 induced into macrophages with PMA and LPS combined for the AS inflammation model.The best intervention concentrations of Simvastatin,Brazilin and Shenxiaheji were determined by CCK-8 experiment.The experiment is divided into five groups,which were control group,model group,simvastatin group,Brazilin group and Shenxiaheji group.Testing the expression of PD-1and related factors by flow cytometry.Testing the expression of PD-1,TLR-4,NF-?B and MMP-9 mRNA in different groups by the qRT-PCR.Testing the concentration of TNF-? and IL-10 cytokines by elisa.Testing the proteins expression of PD-1,TLR-4,NF-?B and MMP-9 by Western Blot.All of above experiments were to reveal the effect mechanism of Shenxiaheji and Brazilin on THP-1 cell inflammation.3.Animal experiment: 10 male health C57BL/6 mice were grouped control group.50 male health Apo E knockout C57BL/6 mice were grouped by random number,and divided into model group,simvastatin group,Brazilin and Shenxiaheji group.The Control group and the other 5 groups were given normal ordinary feed and high fat feed,and the latter was used to copy the AS Model.Simvastatin group(10mg/kg/d),Brazilin group(30mg/kg/d),Shenxiaheji group(13g/kg/d)and anti-PD-1 group(5mg/kg/d)were given by intragastric injection and intraperitoneal injection respectively.Control group and Model group mice were given equal volume of normal saline intervention.After 8 weeks of treatment,body weight change,blood lipid level and PD-1 level of mice in each group were detected,and the degree of AS was detected by histomorphology such as HE staining and gross oil red O staining,and the expression intensity was detected by immunohistochemistry.Results: 1.Clinical study results: There was no significant difference in age and gender between the two groups;There were significant differences in serum lipids and hypersensitive C-reactive protein(P<0.05),and there was no statistically significant difference between groups before treatment;Both Shenxiaheji and Simvastatin reduced the expression level of PD-1 on cell surface,and the difference was statistically significant(P<0.05),there was no statistically significant difference between groups before treatment,but there was statistically significant difference between groups after treatment(P<0.05).Before and after treatment,there were statistically significant differences in the thickness,plaque area of carotid artery,TCM syndrome integral and clinical effect after treatment(P<0.05);There was no significant difference between the test group and the Control group before treatment,but there was significant difference after treatment(P<0.05).2.Cell experiment results: The effect of drugs on THP-1 cells survival rate was measured by CCK-8 method after cell culture to logarithmic growth stage.The results showed that the best concentration of Shenxiaheji was 38.8 mg/m L,and the best concentration of Brazilin was 12?g/m L.When the concentration was greater than 18?g/ m L,the cell survival rate did not change much.The best concentration of Simvastatin was 15?g/ m L,and the growth was concentration dependent.Flow cytometry was used to detect the expression of PD-1.Compared with the Model group,the expression of PD-1 on the cell surface in all three groups was reduced.Compared with the Control group,the differences in the Shenxiaheji group,the Brazilin group and the Simvastatin group were significant(P<0.01;P<0.001;P<0.001).TNF-? and IL-10 were detected by Elisa,and the results showed that the concentrations of TNF-? in the drug group was significantly decreased,and compared with the Model group,the three drugs groups had a very significant difference(P<0.001).The differences between Simvastatin group,Shenxiaheji group and the Brazilin group were highly statistically significant(P<0.001).There was no statistical difference between the Brazilin group and the Shenxiaheji group;IL-10 concentration was increased in the drug group,and compared with the Model group,there was a statistical difference in the Simvastatin group,the Shenxiaheji group and the Brazilin group(P<0.001).The comparison between Simvastatin group and Shenxiaheji group showed a statistically significant difference(P<0.001),and had no statistical significance compared with Brazilin group.The expressions of PD-1,NF-?B,TLR-4 and MMP-9 mRNA were detected by qRT-PCR.The results showed that,compared with the Model group,except the effect of Shenxiaheji on PD-1mRNA was statistically significant,the difference of other test groups was extremely significant.There was no significant difference in PD-1mRNA between the test groups,but the difference in MMP-9mRNA between Shenxiaheji group and the Simvastatin group was very significant(P<0.001),and there was significant difference between Shenxiaheji group and brazilin group(P<0.01),and the difference between simvastatin group and brazilin group was statistically significant(P<0.05).The TLR-4mRNA in Shenxiaheji group was significantly different from Simvastatin group and Brazilin group(P<0.001),and there was no significant difference between the Simvastatin group and the Brazilin group.The expressions of PD-1,NF-?B,TLR-4 and MMP-9 protein were detected by Westernblot.Compared with Model group,the expression of MMP-9 in addition to Shenxiaheji group was significantly different(P<0.001),and other drug groups had significantly differences in protein expression(P<0.001).The differences of PD-1 and MMP-9 among different experimental groups were highly statistically significant(P<0.001).The expression of NF-?B in Simvastatin group was significantly different from that in Shenxiaheji group and Brazilin group(P<0.001),and the expression of TLR-4 in Simvastatin group was significantly different from that in Shenxiaheji group and Brazilin group(P<0.001),and there was no significant statistical difference between Shenxiaheji group and Brazilin group(P>0.05).3.Animal experiments: Compared with the Control group,LDL-C,TG and TC in serum of mice in the Model group were significantly increased,while HDL-C was significantly decreased.AP was obvious in the model group,and there were different degrees of stenosis in the arterial lumen,and the elastic plate of the inner media of the artery was fractured.Compared with Model group,to participate in the Shenxiaheji group,Brazilin group and Simvastatin group significantly lower blood lipid level,and the degree of AS in three groups is light,but the blood lipid level and the degree of AS in PD-1 antibody group were worsened.Immunohistochemistry showed that compared with Model group,to participate in the Shenxiaheji group,Brazilin group and Simvastatin group obviously increased concentrations of PD-1 point in the expression of antibodies against PD-1 set of PD-1 point gather expression decreased significantly,prompting that negative resistance to AS of Shenxiaheji and Brazilin has significant correlation with the negative immune regulation.Flow flow results showed that the difference between the Model group and the Control group was statistically significant,indicating that the AS model of Apo E mice was successfully replicated.Compared with the Model group,the expressions of CD4+T,CD8+T,macrophages and PD-1 in the Shenxiaheji group,the Brazilin group and the Simvastatin group were all decreased,and the differences were significant,while the expressions of CD4+T,CD8+T,macrophages and PD-1 in the anti-PD-1 group were increased,and the differences were significant.Conclusions: 1.Shenxiaheji can effectively regulate the immune response of AS patients,and improve clinical syndromes and prognosis with high clinical safety.2.Shenxiaheji and Brazilin can inhibit the abnormal activation of PD-1,TLR-4,NF-?B and MMP-9,which is a good anti-AS drug that can not only reduce blood lipid,but also have immune regulation and anti-inflammatory activity,laying a solid theoretical foundation for future screening and innovation of anti-AS drugs.3.The theory of "microcosm accumulation" has a very good guiding role in the treatment of AS with Shenxiaheji and Brazilin,which can more effectively combine tumor diseases with the treatment of AS,and provide ideas and directions for solving the two major disease groups in the current world. |
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