Study On Heat-toxic Syndrome Of Unstable Angina And Mechanism Of Anti-atherosclerosis With The Method Of Replenishing Qi And Detoxification

Objective:1.Clinical research:Collecting clinical data,screening and analyzing the elements that may reveal the heat-toxic syndrome,and exploring the relationship between the inflammatory mechanism and the heat-toxic syndrome in heart disease,in order to achieve the objectification and standardization of heat-toxic syndrome differentiation.2.Experimental research:According to the heat-toxic theory of heart disease mentioned by Ding Shuwen,a famous Chinese medicine practitioner in China,and on the basis of previous proof that Chinese herbs for replenishing qi and detoxification could reduce the inflammatory response of cardiovascular diseases,it was proved that as the representative medicine of"the method of replenishing qi,activating blood and detoxification",Astragali Radix–Coptis Rhizoma herb pair could maintain the balance of M1/M2 macrophages and Th1/Th2 lymphocytes in atherosclerotic plaques,inhibit the inflammatory response in plaques,reduce plaque vulnerability,and play an anti-AS role.The inhibitor of signal transducer and activator of transcription 6(STAT6)was given to prove that the regulation of macrophage and T lymphocyte differentiation by Astragali Radix–Coptis Rhizoma herb pair was related to its activation of STAT6 signal pathway.Methods:1.Clinical study:The UA patients who met the criteria were included and divided into heat-toxic syndrome group and non-heat-toxic syndrome group according to the TCM diagnostic criteria.The medical history,blood lipids,inflammatory factors and other clinical data of the patients in the two groups were collected and analyzed,and the two-category logistic regression analysis was performed on the indicators that may reveal the etiology of heat-toxic syndrome.2.Experimental study:(1)8-week-old Apo E-/-mice were divided into 5 groups,and the AS model was established by high-fat diet.The groups were divided into:model control group,Astragali Radix group,Coptis Rhizoma group,Astragali Radix–Coptis Rhizoma group,and statin group.A blank control group was set up,and C57/BL mice of the same age were selected and fed normally.From the 13th week,the corresponding drugs in each group were given by gavage for 6 weeks.Monitor body weight continuously.Serum triglyceride(TG)and cholesterol(TC)levels were compared in each group.Oil red O staining was used to observe the lipid deposition content in aortic plaques.The expression of mononuclear macrophages(MOMA-2)was observed by immunohistochemical staining to reflect the content of macrophages in plaques.The expression ofα-smooth muscle actin(α-SMA)was observed by immunohistochemical staining to reflect the number of vascular smooth muscle cells.Sirius red staining was used to observe the collagen area of the plaques.Plaque vulnerability index(VI)=(lipid content in plaque%+macrophage content%)/(collagen content%+smooth muscle cell content%).Elisa was used to detect the expressions of serum matrix metalloproteinase-9(MMP-9),pro-inflammatory factors interleukin-12(IL-12)and interferon-γ(IFN-γ),anti-inflammatory factors IL-10 and IL-4.Western blot detected the content of aortic M1macrophage marker inducible nitric oxide synthase(i NOS)and M2 macrophage marker arginase-1(Arg-1),Th1 lymphocyte marker T cells Expression of T-box(T-bet)and Th2lymphocyte marker GATA-binding protein 3(GATA-3).Double antibody immunofluorescence detected the positive expression of aortic M1/M2 macrophage markers and Th1/Th2 lymphocyte markers.The spleens of mice were taken,and the numbers of M1 and M2 macrophages,Th1 and Th2 lymphocytes in each group were detected by flow cytometry.(2)8-week-old Apo E-/-mice were given a high-fat diet and were divided into model control group,Astragali Radix–Coptis Rhizoma group and Astragali Radix–Coptis Rhizoma+STAT6 inhibitor(AS1517499)group.In addition,C57/BL mice of the same age were set as blank control group,which was fed with normal food.From the 13th week,the corresponding drug treatment in each group was given for 6weeks.The mice in the Astragali Radix–Coptis Rhizoma group and the Astragali Radix–Coptis Rhizoma+AS1517499 group were given Astragali Radix–Coptis Rhizoma decoction(5.2 g/Kg/day)by gavage,and the Astragali Radix–Coptis Rhizoma+AS1517499 group was given intraperitoneal injection of STAT6 inhibitor AS1517499(10mg/kg/day).Western blot was used to detect the levels of STAT6 and p-STAT6 in the aorta,and the other indicators were the same as(1).Results:1.Clinical research:A total of 319 UA patients were included,40.4%patients were with heat-toxic syndrome,and 59.6%patients were with non-heat-toxic syndrome.The distribution of TCM syndrome types showed that there were many excess syndromes,deficiency and excess mixed syndromes,and the frequency of heat toxin and blood stasis syndrome,qi deficiency and blood stasis syndrome were high.Smoking and hyperlipidemia may be risk factors for the formation of UA heat-toxic syndrome.There were differences in blood lipids including TC,TG and low-density lipoprotein cholesterol(LDL-C)between the heat-toxic syndrome group and the non-heat-toxic syndrome group.And there was no significant difference in high-density lipoprotein cholesterol(HDL-C)between the two groups.In terms of inflammatory indicators,high-sensitivity C-reactive protein(hs-CRP),IL-6 and pro-inflammatory factor tumor necrosis factor-α(TNF-α)in the heat-toxic syndrome group were significantly higher than those in the non-heat-toxic syndrome group,and anti-inflammatory factors IL-4 was lower than that of the non-heat-toxic syndrome group.LDL-C,hs-CRP and TNF-αwere positively correlated with the occurrence of heat-toxic syndrome in UA,and could be used as independent factors for predicting heat-toxic syndrome.2.Experimental study:(1)Compared with the model group,the body weight and blood lipid metabolism of the mice in each drug intervention group were improved,and the Astragali Radix–Coptis Rhizoma group had more obvious effect of reducing TC and TG than the Astragali Radix and Coptis Rhizoma single-drug groups,but not as small as the statin group.The serum levels of pro-inflammatory factors IL-12 and IFN-γin the mice in each drug intervention group were lower than those in the model group,and the levels of anti-inflammatory factors IL-10 and IL-4 were higher than those in the model group.Among the three groups of traditional Chinese medicine groups,Astragali Radix–Coptis Rhizoma group had the most obvious effect on reducing IL-12,IFN-γand increasing IL-10 and IL-4,but compared with statin group,the effect of statin group was better.Compared with the model group,the aortic plaque vulnerability of the mice in each drug intervention group was reduced,and the statin group had the most obvious effect,while Astragali Radix–Coptis Rhizoma group had the best effect than the single drug group.Compared with the model group,the number of M1 and Th1 cells in the spleen of each drug intervention group decreased,while the number of M2 and Th2 cells increased,and the imbalance of M1/M2 and Th1/Th2ratios was improved.The expression of i NOS and T-bet proteins in the aorta decreased,while the expression of Arg-1 and GATA-3 increased.Double-antibody immunofluorescence showed that the positive expression of MOMA-2~+i NOS~+double-positive marker M1 macrophages and CD4~+T-bet~+double-positive marker Th1lymphocytes in the aorta of each drug intervention group decreased,the positive expression of MOMA-2~+Arg-1~+double positive marker M2 macrophages and CD4~+GATA-3~+double positive marker Th2 lymphocytes increased.Compared with the Astragali Radix group and the Coptis Rhizoma group,the Astragali Radix–Coptis Rhizoma group could significantly reduce the number of M1 and Th1 cells in the spleen,and increase the number of M2 and Th2 cells,thereby improving the imbalance of M1/M2and Th1/Th2 ratios.And the protein expression of i NOS and T-bet in the aorta were more obviously reduced,and the expression of Arg-1 and GATA-3 were increased.At the same time,immunofluorescence showed that the positive expression of MOMA-2~+i NOS~+double-positive marker M1 macrophages and CD4~+T-bet~+double-positive marker Th1lymphocytes decreased significantly,the positive expression of MOMA-2~+Arg-1~+double positive marker M2 macrophages and CD4~+GATA-3~+double positive marker Th2lymphocytes increased.Then the Astragali Radix–Coptis Rhizoma group was compared with the statin group,and the results showed that the improvement effect of the statin group was stronger.It is suggested that the Astragali Radix–Coptis Rhizoma herb pair could improve AS by regulating the balance of M1/M2 macrophages and Th1/Th2lymphocytes.(2)Compared with the model group,Astragali Radix–Coptis Rhizoma herb pair could regulate the imbalance of M1/M2 and Th1/Th2 cells,inhibit the progress of AS and reduce the vulnerability of aortic plaque.At the same time,Astragali Radix–Coptis Rhizoma herb pair treatment could increase the expression of p-STAT6,indicating that Astragali Radix–Coptis Rhizoma herb pair could activate STAT6 pathway.The combined administration of STAT6 inhibitor decreased the immunomodulatory and anti-AS effects of Astragali Radix–Coptis Rhizoma herb pair,indicating that the mechanism of Astragali Radix–Coptis Rhizoma herb pair producing the above effects was related to its activation of STAT6 pathway.Conclusion:1.Clinical research:The investigation on the distribution law of TCM syndrome types in UA found that excess syndrome,deficiency and excess mixed syndrome were more common,among which heat toxin and blood stasis syndrome,qi deficiency and blood stasis syndrome were most distributed.The heat-toxic syndrome plays an important role in the TCM classification of UA.The patients with heat-toxic syndrome in UA were mostly clustered with risk factors such as smoking and hyperlipidemia population,with the increase of pro-inflammatory factors and the decrease of anti-inflammatory factors.LDL-C,hs-CRP and TNF-αwere independent factors for predicting the etiology of UA heat-toxic syndrome.The above results suggest that clinical attention should be paid to patients with coronary heart disease with abnormal blood lipid metabolism and aggravated inflammatory response,timely medicine treatment and publicity and education,control of blood lipid and inflammatory response,and addition of traditional Chinese medicine for replenishing qi,clearing heat and detoxification on the basis of western medicine treatment,to better improve the patient’s health,clinical symptoms and prognosis.2.Experimental study:(1)High-fat diet-induced AS model mice exhibited immune-inflammatory imbalance,increased pro-inflammatory M1-type macrophages and Th1-type lymphocytes,and decreased the number of anti-inflammatory M2-type macrophages and Th2-type lymphocytes,resulting in the imbalance of M1/M2 and Th1/Th2 cells.Compared with a single herb,the Astragali Radix–Coptis Rhizoma herb pair could better regulate the inflammatory response in both directions,improve the immune-inflammatory imbalance of AS,and play a role in the treatment of AS.(2)Astragali Radix–Coptis Rhizoma herb pair has the effect of replenishing qi and detoxifying in the treatment of AS by regulating the imbalance of M1/M2 macrophages and Th1/Th2 lymphocytes,and its mechanism is closely related to the activation of STAT6 pathway.