| Part one:clinical studyObjective:The aim of the first part of study is to investigate the effect of Tongyang Kuanxiong Granule on patients with stable angina pectoris of coronary heart disease score,and to explore the effect on serum lipid and inflammatory cytokines,including interferon gamma（IFN-γ）,interleukin17（IL17）,anti-inflammatory cytokine transforming growth factor beta 1（TGF-β1）,interleukin 10（IL10）,so that to reveale the mechanism of Tongyang Kuanxiong particles on atherosclerosis（AS）.Method:72 patients with stable angina disease was slected and randomly divided into two groups,the treatment group and control group,36 cases in each group.the patients in control group was given oral administration of Atorvastatin Calcium Tablets,the patients in treatment group were additionally gaven Tongyang Kuanxiong granules.After 2 months of treatment,patients were evaluated the TCM symptom score,serum lipid levels,and the serum content of interferon gamma（IFN-γ）,interleukin 17（IL17）,transforming growth factor beta 1（TGF-β1）,interleukin 10（IL10）.Result:（1）63 patients have completed the clinical study.4 cases in treatment group and 5 cases in control group have lost to follow up,therefore,32cases intreatment group and 31case in coutrol group have completed the study.The result shows that,before treatment,there were no statisticant significance in gender,age,TCM symptom score,total cholesterol（TC）,serum triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）,serum transforming growth factor beta 1（TGF-β1）,interferon gamma（INF-γ）,interleukin-10（IL-10）,interleukin-17（IL-17）levels between groups（P<0.05）.（2）There are statisticant significance in TCM symptom scores when make comparation before and after treatment in each group（P<0.05）.The decrease of TCM symptom scores and the increase of the effective rate in treatmeat group were significantly different compared with that in the control group（P<0.05）.（3）There are statisticant significance in angina pectoris scores when make comparation before and after treatment in each group（P<0.05）.The decrease of angina pectoris scores and the increase of the effective rate in treatmeat group were significantly different compared with that in the control group（P<0.05）.（4）There are statisticant significance in TC,TG,LDL-C and HDL-C levels when make comparation before and after treatment in each group（P<0.05）.The increase in HDL-C level and the deacease in TC,TG,LDL-C levels in treatment group were significantly different compared with that in the control group（P<0.05）.（4）There are statisticant significance in TGF-β1,IL-10,INF-γand IL-17 levels when make comparation before and after treatment in each group（P<0.05）.The increase in TGF-β1,IL-10 levels and the deacease in INF-γand IL-17 levels in treatment group were significantly different compared with that in the control group（P<0.05）.Conclusion:（1）Tongyang Kuanxiong granules combine with atorvastatin can improve the clinical symptoms and efficiency of patients with stable angina pectoris effectively.（2）Tongyang Kuanxiong granules combine with atorvastatin can effectively regulate the metabolism of blood lipids,which may play an importment role in retarding the progress of atherosclerosis.（3）Tongyang Kuanxiong granules combine with atorvastatin could promote endogenous anti-inflammatory effects by effectively reducing the inflammation related inflammation factors,including raise TGF-beta1、IL10and reduce Th17、INF-γ,which both were good for improving atherosclerosis.Part two:experimental studyObjective:The aim of the second part of study was to explore the mechanism of Tongyang Kuanxiong particles on atherosclerotic in ApoE-/-mice by testing observing the serum lipid level,the gene expression of MMP2,MMP9,LOX-1,ABCA1,TGF-β1,Smad3,the protein expression of TGF-β1,Smad3,IL10 and Th17 in aorta wall and measuring the regulatory T cells（Treg）level in the spleen of mice,so that to explore the mechanism of Tongyang Kuanxiong particles on lipid transport,immune related intravascular inflammation and plaque stability of atherosclerosis.Methods:12 normal C57BL/6mices were set as control group,Animal models of atherosclerosis were made by high fat diet fed for 60 ApoE-/-mices.Then the 60 ApoE-/-mices were randomly divided into six groups,including model group,atorvastatin group,atorvastatin+Chinese medicine group,low-dose of Chinese medicine group,medium dose of Chinese medicine group and high dose group of Chinese medicine group,with 10 mices with half male and half female in each group.After the atherosclerosis model is completed,the mices in control group and model group were given normal saline by the method of gavage administration（0.025ml/g/day）.Micesin atorvastatin group were given atorvastatin calcium tablets（0.0026mg/g/day）.Mices in Chinese medicine intervention group（low dose group,medium dose group and high dose group）were given the corresponding dose of Chinese medicine（3.12mg/g/day,6.24mg/g/day,12.48mg/g/day）.Mices in Chinese medicine+atorvastatin group was given atorvastatin calcium solution（0.0026mg/g/day）and medium dose of Chinese medicine（6.24mg/g/day）.The interventions were gaven 1 time a day for 12 weeks.After12weeks,miceswereanesthetizedand extracted the eyeball blood as much as possible.Then the TC,TG,LDL-C,HDL-C levels were measured using enzyme immunoassay method.The gene expression level of LOX-1,ABCA1,MMP2,MMP9,TGF-β1 and Smad3 in aortic wall were measured by the method of PCR technique.The protein expression level of TGF-β1,Smad3,IL10 and Th17in aortic wall were measured by immunohistochemistry.The aortic wall was made paraffin sections and stained by oil red O to observe the atherosclerosis formation under the microscope.The fresh spleen of the mices were used to detect the number of regulatory T cells（Treg）by the method of flow cytometry.Results:（1）After 12 weeks of the high-fat diet,the atherosclerotic plaque could successfully form in ApoE-/-mice aortic wall.The intervention with high dose of Tongyang Kuanxiong granule or Tongyang Kuanxiong granules+atorvastatincan significantly inhibit the formation atherosclerotic plaque compared that with model group,atorvastatin group,low dose and medium dose of Chinese medicine group（P<0.05）.（2）The change in serum lipid metabolism:After intervention,compared with the model group,atorvastatin group,low dose and medium dose of Chinese medicine group,high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group can significantly decrease TC,TG and LDL-C levels（P<0.05）and significantly increase the level of HDL-C（P<0.05）.There are no significant difference between high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group in decrease TC,TG and LDL-C and increase HDL-C levels（P>0.05）.（3）The gene expression of LOX-1 in the aortic wall of mice:Compared with the model group,he gene expression of LOX-1 were statistically decreased in Chinese medicine or atorvastatin intervention groups（P<0.05）.Compared with atorvastatin group,low dose and medium dose of Chinese medicine group,the expression of LOX-1 gene in high dose Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group were significantly decreased（P<0.05）.There are no significant difference between high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group in the gene expression of LOX-1（P>0.05）.（4）The gene expression of ABCA1 in the aortic wall of mice:Compared with the model group,atorvastatin group,low dose and medium dose of Chinese medicine group,the expression of ABCA1 gene were significantly increased in the Chinese medicine+atorvastatin group and high dose of Chinese medicine group（P<0.05）.However,there were no significant difference in the expression of ABCA1 gene between the atorvastatin group,the low dose and medium dose of Chinese medicine group（P>0.05）.There were no significant difference in the expression of ABCA1 gene between high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group（P>0.05）.（5）The gene expression of TGF-β1in the aortic wall of mice:Compared with the model group,the expression of TGF-β1 gene were significantly increased in Chinese medicine or atorvastatin intervention groups（P<0.05）.Compared with atorvastatin group,medium dose of Chinese medicine group,the expression of TGF-β1 gene were significantly increased in Chinese medicine+atorvastatin group and high dose Chinese medicine group（P<0.05）.There were no significant difference in the gene expression of TGF-β1 among the high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group（P>0.05）.（6）The gene expression of Smad3 in the aortic wall of mice:Compared with the model group,the expression of Smad3 gene in the aorta of Chinese medicine+atorvastatin group and medium dose and high-dose of Chinese medicine group were significantly increased（P<0.05）.Compared with the atorvastatin group,low dose and medium dose of Chinese medicine group,the expression of Smad3 gene in the Chinese medicine+atorvastatin group and high dose of Chinese medicine group were significantly increased（P<0.05）.There were no significant difference in Smad3 gene expression among the atorvastatin group,the low dose and medium dose of Chinese medicine group（P>0.05）.There were no significant difference in the gene expression of Smad3 among the high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group（P>0.05）.（7）The gene expression of MMP2 in the aortic wall of mice:Compared with the model group,the gene expression of MMP2 in the Chinese medicine+atorvastatin group,medium dose and high dose Chinese medicine group were significantly decreased（P<0.05）.Compared with the atorvastatin group,the gene expression of MMP2 in the high dose Chinese medicine group was significantly decreased（P<0.05）.Compared with the low dose and medium dose of Chinese medicine group,the gene expression of MMP2 in the high dose Chinese medicine group and Chinese medicine+atorvastatin group were significantly decreased（P<0.05）.There were no significant difference in gene expression of MMP2 among the atorvastatin group,the low dose of Chinese medicine group（P>0.05）.There were no significant difference in the gene expression of MMP2 among the high dose of Chinese medicine group and Tongyang Kuanxiong granule+atorvastatin group（P>0.05）.（8）The gene expression of MMP9 in the aortic wall of mice:Compared with the model group,all the drug intervention groups could significantly reduce the gene expression of MMP9 in the aorta of mice（P<0.05）.Compared with the atorvastatin group,low dose and medium dose of Chinese medicine group,the gene expression of MMP9 in the high dose group and Chinese medicine+atorvastatin group were significantly decreased（P<0.05）.There was no significant difference in the gene expression of MMP9 among the atorvastatin group,the low dose andmedium dose of Chinese medicine group（P>0.05）.（9）the content of CD4+CD25+Foxp3+T cells（Treg）in the spleen of mice:Compared with the model group,atorvastatin group,the low dose of Chinese medicine group,the number of CD4+CD25+Foxp3+T cells in mice spleen of Chinese medicine+atorvastatin group,medium dose and high dose of Chinese medicine group were significantly increased（P<0.05）.Compared with medium dose of Chinese medicine group,Chinese medicine+atorvastatin group,the number of CD4+CD25+Foxp3+T cells in mice spleen of high-dose of Chinese medicine were significantly increased（P<0.05）.There was no significant difference in the number of CD4+CD25+Foxp3+T cells in mice spleen of model group,atorvastatin group,the low dose of Chinese medicine group（P>0.05）.（10）The immunohistochemistry detection of TFG-β1 in mice aorta:Compared with the model group,the protein expression of TFG-β1 in Chinese medicine+atorvastatin group,low dose,medium dose and high dose of Chinese medicine group were significantly increased（P<0.05）.Compared with atorvastatin group,the protein expression of TFG-β1 in Chinese medicine+atorvastatin group,medium dose and high dose of Chinese medicine group were significantly increased（P<0.05）.Compared with the low dose,medium dose of Chinese medicine group,the protein expression of TFG-β1in Chinese medicine+atorvastatin group,high dose of Chinese medicine group were significantly increased（P<0.05）.There was no significant difference in the protein expression of TFG-β1 between Chinese medicine+atorvastatin group and high dose of Chinese medicine group（P>0.05）.（11）The immunohistochemistry detection of Smad3 in mice aorta:Compared with the model group,the protein expression of Smad3 in drug intervention groups were both significantly increased（P<0.05）;Compared with atorvastatin group and low dose of Chinese medicine group,the protein expression of Smad3 in Chinese medicine+atorvastatin group,medium doseand high dose of Chinese medicine group were significantly increased（P<0.05）.Compared with medium dose of Chinese medicine group,the protein expression of Smad3 in Chinese medicine+atorvastatin group and high dose of Chinese medicine group were significantly increased（P<0.05）.There were no significant difference in the protein expression of Smad3 between Chinese medicine+atorvastatin group and high dose of Chinese medicine group（P>0.05）.（12）The immunohistochemistry detection of IL10 in mice aorta:Compared with the model group and low dose of Chinese medicine group,the protein expression of IL10 in Chinese medicine+atorvastatin group and high dose of Chinese medicine group were both significantly increased（P<0.05）;Compared with atorvastatin group and medium dose of Chinese medicine group,the protein expression of IL10 in high dose of Chinese medicine group was significantly increased（P<0.05）.There were no significant difference in the protein expression of IL10 between atorvastatin group,low dose and medium dose of Chinese medicine group（P>0.05）.There were no significant difference in the protein expression of IL10 between Chinese medicine+atorvastatin group and high dose of Chinese medicine group（P>0.05）.（13）The immunohistochemistry detection of IL17 in mice aorta:Compared with the model group,atorvastatin group and low dose and medium dose of Chinese medicine group,the protein expression of IL17 in Chinese medicine+atorvastatin group and high dose of Chinese medicine group were both significantly decreased（P<0.05）;Compared with atorvastatin group andlow dose of Chinese medicine group,the protein expression of IL17 in Chinese medicine+atorvastatin group,medium dose and high dose of Chinese medicine group were significantly decreased（P<0.05）.There were no significant difference in the protein expression of IL17 between atorvastatin group and low dose of Chinese medicine group（P>0.05）.There were no significant difference in the protein expression of IL17 between Chinese medicine+atorvastatin group and high dose of Chinese medicine group（P>0.05）.Conclusion:（1）The application of Tongyang Kuanxiong granules or Tongyang Kuanxiong+atorvastatin can both effectively regulate the blood lipid transport level,inhibit the lipid accumulation of atherosclerotic plaque in ApoE-/-atherosclerotic mice.（2）The intervention of high dose Tongyang Kuanxiong granules or Tongyang Kuanxiong+atorvastatin can significantly improve the expression of TGF-β1/Smad3 pathway,which could improve the inflammatory reaction state of atherosclerosis.（3）The intervention of medium and high dose of TongYang Kuanxiong granules can significantly regulate the Treg/Th17 blance,which effect could interact with the TGF-β1/Smad3 pathway and improve the inflammatory reaction of atherosclerosiscooperately.（4）The intervention of high dose of Tongyang Kuanxiong granules can effectively reduce the gene expression levels of MMP2 and MMP9 in ApoE-/-atherosclerotic mice,which effect could be regulated by the TGF-β1/Smad3pathway and improve the stability of atherosclerotic plaques.（5）There was a dose effect relationship of TongYang Kuanxiong granules in the effect of improving lipid levels,regulating lipid transport balance,inhibiting of plaque lipid deposition,regulating inflammatory response,regulate the balance of Treg/Th17,improving the stability of plaque in ApoE-/-mice.And the high dose of TongYang Kuanxiong granules was recommended for atherosclerosis. |
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