Experimental Study Of Heat-Clearing,Blood-Activating Traditional Chinese Drug Regulating The TLR4 Signaling Pathway On Atherosclerosis Rats And The Clinical Study Of The Cause Of The Toxic Heat Syndrome On Acute Myocardial Infarction

Part 1 Experimental study on the effects of chronic inflammation induced by LPS in the development of atherosclerosis ratsObjectiveWe aim to investigate the possible mechanism and the effects of chronic inflammation induced by LPS in the development of atherosclerosis rats in this experiment. On the basis of high fat diet combined with intraperitoneal injection of vitamin D3, we intramuscularly inject different dose of LPS to cause a chronic inflammation in the rats. By three methods to develop the atherosclerosis rats model, we observe the development of atherosclerotic plaque and the blood lipid and inflammatory factors caused by LPS in the rats, in order to investigate the method and the possible mechanism of a fast, stable and economical atherosclerosis rat modelMethodTo construct the AS model by the way of imitating the chronic inflammation process by injecting LPS intramuscularly with high fat diet combined with intraperitoneal injection of vitamin D3.32 Male SD rats, SPF level, weighing from 180-220g were used to divided into 4 groups according to the different interventions, normal group(normal diet),model group(high fat diet combined with intraperitoneal injection of vitamin D3),low dose group(injecting low dose of LPS intramuscularly with high fat diet combined with intraperitoneal injection of vitamin D3), high dose group (injecting high dose of LPS intramuscularly with high fat diet combined with intraperitoneal injection of vitamin D3). Ten weeks after the end of the experiment, the animals were killed by abdominal aortic blood sampling, then we used ELISA method to detect the serum levels of inflammatory factors (TNF-α、IL-6);used enzyme colorimetric method to detect the blood lipid levels (CHOL、TG、HDL-C、 LDL-C);carry on histopathological observation and integral evaluation with HE staining aortic tissue.Results1.The level of TC, TG, LDL-C on the intervention group and the model group were significantly higher than the normal group(P<0.05), which mean the hyperlipidemia rat model was developed. The difference of the blood lipid level between the low dose group and model group were not significant. The level of TC, TG, LDL-C on the high dose group was significantly higher than the model group (R0.05)2. The level of TNF-α、IL-6 on the intervention group and the model group were significantly higher than the normal group(P<0.05), which mean the rats were under the chronic inflammation induced by LPS on the basis of high fat diet combined with intraperitoneal injection of vitamin D3. The level of TNF-a, IL-6 on the high dose group was significantly higher than the model group(P<0.05) 3. The HE staining of the model group show that the structure of the vascular endothelium was disordered, intimal thickening, atherosclerotic plaque, lipid plaque, foam cell.calcium deposition could be seen in the aortic wall, and the atherosclerotic plaque cause luminal narrowing. The low dose group show that the intimal was thickening more seriously, foam cell and the inflammatory cells could be seen in the intimal, more atherosclerotic plaque could be seen in the aortic wall. The high dose group show the most severe disorder in the aorta, the structure of the vascular endothelium was disordered, intimal thickening, a large number of lipid plaque, foam cell, calcium deposition could be seen in the aortic wall, the inflammatory cells could be seen in the intimal, atherosclerotic plaques become unstable. The grade of aortic lesion score in the high dose group was significantly higher than the model group(P<0.05).Conclusion1. The intervention of high fat diet combined with intraperitoneal injection of vitamin D3 could increase the level of TC, TG, LDL-C in the rats. By injecting different dose of LPS intramuscularly could increase the level of TC, TG, LDL-C synergistically. The high dose of LPS cause dyslipidemia more severe than the low dose of LPS.2. The intervention of high fat diet combined with intraperitoneal injection of vitamin D3 could increase the level of TNF-a, IL-6 in the rats. By injecting different dose of LPS intramuscularly could increase the process of inflammatory reaction and the level of TNF-a, IL-6 synergistically. The high dose of LPS could increase the process of inflammatory reaction.3. The possible mechanisms of LPS promoting rats atherosclerosis include the following aspects such as inflammatory reaction and dyslipidemia, releasing the inflammatory corpuscle cause endothelial damage and the foam cells phagocytize lipid, which may increase lipid oxidative metabolism disorder and lipid deposition to promote the process of atherosclerosis and vulnerable plaque.Part 2 Experimental Study of Heat-Clearing, Blood-Activating Traditional Chinese Drug Regulating the TLR4 Signaling Pathway, blood liquid and inflammatory factor on Atherosclerosis RatsObjectiveTo construct the AS model by the way of injecting LPS intramuscularly with high fat diet combined with intraperitoneal injection of vitamin D3. We used the heat-clearing traditional Chinese drug Shuanghuanglian injection and blood-activating traditional Chinese drug Salvianolate injection to treat the atherosclerosis rats induced by complex factors. The outcome of blood lipid, TNF-a、IL-6, P-selectin and the HE staining of aortic pathological morphology were used to evaluate therapeutic effect, and the TLR4/MyD88/NF-kappa B signaling pathway were used to explore the possible mechanism of the intervention on atherosclerosis rats by the shuanghuanglian injection and salvianolate injection, which could provide the basis for heat-clearing method and blood-activating method.Method48 male SD rats(SPF level,180-220g) were divided into 6 groups, the specific grouping was the salvianolate low dose group (DSL,20mg/kg), the salvianolate high dose group (DSH,60mg/kg), the shuanghuanglian low dose group (SHLL,750mg/kg), the shuanghuanglian high dose group (SHLH,1500mg/kg), the model control group and the blank control group. The modeling method is based on the H group in experiment part 1, starting from the sixth week of modeling, intervention by intraperitoneal injection of shuanghuanglian and salvianolate,1 times a day for 6 weeks, both blank control group and model control group were given the same amount of normal saline by intraperitoneal injection. Twelfth weeks after the end of the experiment, the animals were killed by abdominal aortic blood sampling, after the routine fasting water for 12 hours, we make corresponding treatment so that the blood samples were collected, the samples were drawn and the indexes were detected. As before we used ELISA method to detect the serum levels of TNF-α、IL-6、P-selection;used enzyme colorimetric method to detect the blood lipid levels (CHOL、TG、HDL-C、 LDL-C);carry on histopathological observation with HE staining aortic tissue;used immunohistochemistry method and western blot method to detect the expression and level of TLR4, MyD88, and NF-kB in aortic tissue.Results1.The shuanghuanglian injection and salvianolate injection could decrease blood lipid level in different degree. Both the group of shuanghuanglian injection and salvianolate injection could decrease the CHOL, TG, LDL-C level more significantly than the model group(P<0.05). The DSL could decrease the TG level more significantly than SHLL(P<0.05). The LDL-C level of DSH was lower than SHLH, but there was not statistically significant (P>0.05).2. The shuanghuanglian injection and salvianolate injection could blood lipid level of TNF-a, IL-6, P-selectin in different degree. The TNF-a level of the SHLL and DSL were lower than model group, but there was not statistically significant (P>0.05), while the TNF-a level of the SHLH and DSH were lower than model group with statistically significant (P<0.05). The IL-6 level of the SHLL was lower than model group, but there was not statistically significant (P>0.05), while the IL-6 level of the SHLH and DSL, DSH were lower than model group with statistically significant (P<0.05). The P-selectin level of the SHLL and SHLH were lower than model group, but there was not statistically significant (P>0.05), while the P-selectin level of the DSL and DSH were lower than model group with statistically significant(P<0.05).3. Detection of TLR4, MyD88, NF-kappa B protein in aortic tissue by immunohistochemistry and WB method, there is no formation of aortic plaque in blank control group, expression of TLR4, MyD88, NF-kappa B protein in aortic tissue were negative, but in model group, expression of TLR4, MyD88, NF-kappa B protein in aortic plaque showed strong positive; Using different doses of shuanghuanglian injection, salvianolate injection after intervention, significantly reduced expression of TLR4, MyD88, NF-kappa B protein in the plaque, low dose of shuanghuanglian and salvianolate group and model group were decreased atherosclerotic plaque tissue in TLR4, MyD88 protein expression, but the difference was not statistically significant (P>0.05), compared with the model group, high dose of shuanghuanglian and salvianolate group significantly decreased the expression of TLR4, MyD88, and the high dose group than in low dose group (P<0.05); both high and low dose of shuanghuanglian and salvianolate group decreased the expression of NF-kappa B protein compared with the model group (P<0.05), and the high dose group is better than the low dose group (P<0.05).4. Aortic tissue with HE staining under light microscope observation, in the model group, we can see vascular endothelial continuity damaged, the intimal thickening and smooth muscle cell proliferation and migration is visible, multiple plaque formation, lipid plaque and foam cell accumulation, blue granular calcification, inflammatory cells accumulation, we found in plaque rupture, mixed thrombus, aortic lumen stenosis; Low dose of shuanghuanglian injection group in aortic plaque improvement is not obvious than in the model group, from the degree of intimal thickening, foam cells, inflammatory cell infiltration, lipid deposition and plaque size, high dose of shuanghuanglian group and low dose, high dose of salvianolate group were improved than in model group, which mean shuanghuanglian injection, salvianolate injection can preserve the vascular endothelial and reduce the degree of foam cells infiltration and lipid deposition, and then stable the atherosclerosis plaque. The high dose salvianolate group might be better than the high dose shuanghuanglian group in the inhibition of atherosclerosis process.Conclusion1. The expression of TLR4, MyD88, NF-kappa B protein and the level of TNF-a, IL-6 in the atherosclerosis rats induced by complex factors were higher than the normal group, which mean that the high fat diet and LPS stimulus may activate TLR4/MyD88/NF-kappa B signaling pathway and then advance the inflammatory factor downstream to promote the process of atherosclerosis.2. Shuanghuanglian injection, salvianolate injection can reduce the TNF-a, IL-6 level on AS rats induced by composite factors(P<0.05), reduce inflammation, inhibit AS progression, salvianolate can also inhibit Ps to further stabilize atherosclerotic plaque, delaying the progress of it;3. Shuanghuanglian injection, salvianolate injection can reduce the TC、TG、 LDL-C level on AS rats induced by composite factors, in which the salvianolate decreased LDL-C, TG may be better than the effect of shuanghuanglian Injection;4. Shuanghuanglian injection, salvianolate injection may be through the intervention of TLR4/MyD88/NF-κβ signaling pathway by different degree, so that inhibit the development of atherosclerosis, Improving plaque vulnerability, promoting the stability of atherosclerotic plaque;5. Salvianolate may be better than that of shuanghuanglian on AS resistance in rats model induced by composite factors, this is related to that the salvianolate in reducing LDL-C, TG level, inhibiting platelet activating Ps is better than that of shuanghuanglian and other factors.Part 3 Clinical Study of the Cause of the Toxic Heat syndrome on Acute Myocardial InfarctionObjectiveAcute myocardium infarction patients were enrolled in this clinical study. We collected and analyses the AMI patients’data including TCM syndrome, coronary heart disease risk factors, laboratory examination, situation of vascular lesions.hospital courses and complication during the recent years in our hospital, focusing on how to select risk factors that may reveal toxic heat syndrome on AMI by univariate analysis and multiple logistic regression analysis, in order to investigate the pathogenesis of toxic heat syndrome on AMI and provide clinical evidence of using the method of heat-clearing, blood-activating to treat the coronary heart disease.MethodBy single center, cross-sectional, retrospective survey method, the AMI patients who meet the inclusion criteria as the research object, design <Coronary Heart Disease Patients With Acute Myocardial Infarction-Syndrome and Condition Questionnaire), collect and collate the patients’Chinese medicine syndrome, coronary heart disease risk factors, physical and chemical examination, hospital condition and other information, focus on how to select risk factors that may reveal toxic heat syndrome on AMI by univariate analysis and multiple logistic regression analysis.Results1. There were 322 patients enrolled in this study. In the distribution of TCM symptoms on AMI patients, excess symptoms and symptoms of intermingled deficiency and excess are more than deficient symptoms. The frequency distribution of syndromes are mostly concerning the blood stasis, heat toxin, sputum cloud and qi stagnation syndrome in the main excess symptoms. Among the excess syndrome, the most common is heat toxin and blood stasis, sputum and blood stasis. In the syndrome of intermingled deficiency and excess, blood stasis, sputum and qi deficiency are the highest proportion. According to the data collected from a retrospective survey in our hospital, the order of frequency in the AMI patients is heat toxin 42.20%(136 patients), non-heat toxin 57.8%(186 patients).2.In this study, male was at high risk in AMI patient with the proportion of 82.9%. The proportion of smoking, hypertension, diabetes, hyperlipidemia, cardiovascular diseases family history were 37.2%,51.8%,36.6%,64.9%,12.4%. According to the univariate analysis, toxic heat syndrome in the AMI patients are the multiple cardiovascular risk factors people with smoking, spicy and high-fat diet, diabetes, hyperlipidemia (P<0.05). Smoking, low-density lipoprotein and triglyceride are the independent risk factors according to the multiple logistic regression analysis and the odds ratio were 1.755,1.637,1.483.3. Prognosis between the toxic heat syndrome and non-toxic heat syndrome, the number of the culprit vessel and triple vessel in the heat toxin AMI patients is higher than non-heat toxin, which suggest that the toxic heat syndrome group of coronary artery lesion is more severe than in non-toxic heat syndrome group and the difference is statistically significant (P<0.05). There are no significant difference in the distribution between groups in the PCI complications such as pump function, low blood pressure, mechanical complications, pulmonary infection(P>0.05), except the malignant arrhythmia, the incidence rate is higher in non-toxic heat syndrome group(P<0.05). In the index of left ventricular ejection fraction and left ventricular diastolic volume by echocardiographic examination, the group of heat toxin is more severe than the group of non-heat toxin (P<0.01), which suggest that the group of heat toxin in AMI patients may have a poor long-term outcome with major adverse cardiovascular events. The rigorous conclusions need to be draw by a complete follow-up survey.ConclusionIn the distribution of TCM symptoms on AMI patients, excess symptoms and symptoms of intermingled deficiency and excess are more than deficient symptoms. The frequency distribution of syndromes are mostly concerning the blood stasis, heat toxin and sputum cloud syndrome in the main excess symptoms. According to the univariate analysis, toxic heat syndrome in the AMI patients are the multiple cardiovascular risk factors people with smoking, spicy and high-fat diet, diabetes, hyperlipidemia. Smoking, low-density lipoprotein and triglyceride are the independent risk factors according to the multiple logistic regression analysis.The toxic heat syndrome in AMI patients are more severe than in non-toxic heat syndrome group in the coronary artery lesion and left ventricular ejection fraction and left ventricular diastolic volume, which suggest that the group of toxic heat syndrome in AMI patients may have a poor long-term outcome with major adverse cardiovascular events. The coronary heart disease patients with long-term smoking, hyperlipidemia should be strengthened to give up smoking and improve the dietary pattern, together with reduce blood lipid levels. On the basis of medicine treatment, we should increase the Chinese medicinal formulae of heat-clearing and blood-activating in order to improve patient’s clinical symptoms and long-term prognosis.