Protective Mechanism And Application Research Of Compound QiMa Capsule On Vascular Endothelial Dysfunction In Essential Hypertension

Part Ⅰ Clinical studyObjectiveTo investigate effect of the compound QiMa capsule on blood pressure, clinical symptoms, endothelial function and endothelial injury factors in essential hypertension patients who discriminated as qi deficiency and sputum turbid type, to evaluate the efficacy of the compound QiMa capsule on endothelial function.Methods80 patients with essential hypertension and discriminated as qi deficiency and sputum turbid type were enrolled, and then ramdomly divided into control group and therapy group. Control group received oral Nifedipine, and therapy group received Nifedipine and compound QiMa capsule, for 8 weeks’ therapy respectively. Then observed the patients’ blood pressure, clinical symptoms, serum angiotensin Ⅱ, endothelial microparticles, brachial artery flow mediated of diastolic function before and after therapy.Results(1)74 patients completed the research, the control group 36, the therapy group 38, two groups of patients before treatment in gender, ages, course of the disease, the grade of hypertension, Chinese medicine symptom integral, AngⅡ, EMPs and FMD levels, there was no statistically significant difference (P>0.05).(2) Comparison of symptom score of TCM after treatment. There were significant reduction in the symptom score of TCM(P<0.05). The symptom score of TCM is significantly higher than control group(P<0.05).(3)Comparison of antihypertensive effects in two groups after treatment. Two groups of SBP and DBP was a significant reduction after treatment (P <0.05). Treatment group before and after treatment in patients with SBP difference is significantly higher than control group(P<0.05), DBP decreased, there was no significant difference(P>0.05).(4)Comparison of the level of FMD in two groups after treatment. Two groups of FMD levels were significantly increased than before treatment(P<0.05), but the treatment group had a significantly higher degree of FMD levels rise in the control group(P<0.05).(5)Comparison of the level of serum AngⅡ in two groups after treatment. There was no significant change in control group (P>0.05). The treatment group of Ang II level was decreased significantly and the drop degree was also significantly higher than that of control group(P<0.05).(6)Comparison of the plasma level of EMPs in two groups after treatment. Two groups of EMPs level was decreased significantly, the difference was statistically significant (P<0.05), but the degree of a decrease in the level of EMPs treatment group is significantly higher than control group(P<0.05). Conclusions(1)The compound of QiMa capsule can not only reduce blood pressure, but also can improve clinical symptoms of essential hypertension patients who discriminated as qi deficiency and sputum turbid type.(2)The compound of QiMa capsule may reduce the level of EMPs and AngⅡ in order to protect the endothelial function.Part II Experiment researchExperiment I Study on the mechanism of Compound QiMa capsule on vascular endothelial dysfunction in Essential hypertensionObjectiveTo observe the effective of the compound of QiMa capsule on blood pressure, vascular endothelial active substance (NO, ET 1 and Ang Ⅱ) and oxidative stress related factor (SOD and MDA) in spontaneously hypertensive rats and to explore the mechanisms of the compound of QiMa capsule restores essential hypertension endothelial dysfunction.Methods50 spontaneously hypertensive rats (SHR) aged 14 weeks were randomly divided into 5 groups, SHR group (given distilled water,10 ml/Kg/d), Nifedipine group (Nifedipine,2.7 mg/Kg/d), Nifedipine combine compound QiMa capsule group (nifedipine,2.7 mg/Kg/d+compound QiMa capsule,0.9288 g/Kg/d), compound QiMa capsule high-dose group (compound QiMa capsule,3.7125 g/Kg/d), compound QiMa capsule low dose group (compound QiMa capsule,0.9288 g/Kg/d), only with 10 WKY rats as normal controls (distilled water,10 ml/Kg/d). The blood pressure of 6 groups were taken before the first delivery and every 2 weeks after the treatment respectively using noninvasive tail artery manometry, delivery time for eight weeks.After eight weeks, the blood sample was taken from abdominal aortic and tested the serum NO, ET-1, Ang Ⅱ, SOD, MDA. Taken 1 cm aorta tissue for HE staining and then observed its morphology under the microscope observation. To test the aorta tissue expression of mRNA and protein level of eNOS, NOX4 and Sirtl.Result(1)After 2 weeks, Nifedipine group of SBP and DBP level was significantly lower than that of SHR group (P<0.05), Nifedipine combine compound QiMa capsule group of SBP was also significantly lower than that of SHR group(P<0.05). Since 4 weeks, the rest group of SBP and DBP were lower than that of SHR group, the difference was statistically significant (P<0.01 or P<0.05). Compared with before treatment, the Nifedipine group and Nifedipine combine compound QiMa capsule group of SBP and DBP significantly decline after two weeks, the difference was statistically significant(P<0.01). Compound QiMa capsule high-dose group of SBP and DBP was decreased significantly(P<0.01) after 4 weeks, while Compound QiMa capsule low-dose group of SBP and DBP was decreased significantly(P<0.01) after 8 weeks.(2)The comparison of serum NO in each group. Compared with WKY group, the other groups of serum NO level were decreased obviously (P<0.01). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of the serum NO level were significantly increased (P<0.01). Compared with nifedipine group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of the serum NO level were significantly increased (P<0.05). But there were no significant among Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group.(3) The comparison of serum ET-1 in each group. Compared with WKY group, SHR groups of serum ET-1 level was increased obviously, the difference was statistically significant(P<0.05). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of the serum ET-1 level were significantly decreased (P<0.05). There were no significant among Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group.(4) The comparison of serum AngⅡ in each group. Compared with WKY group, SHR groups of serum AngⅡ level was increased obviously, the difference was statistically significant(P<0.01). Compared with SHR group, Nifedipine, Nifedipine combine compound QiMa capsule and Compound QiMa capsule high-dose group level of the serum AngⅡ were significantly decreased(P<0.05). There were no significant between Compound QiMa capsule low-dose group and SHR group (P>0.05).(5) The comparison of serum MDA in each group. Compared with WKY group, SHR groups the level of serum MDA was increased obviously, the difference was statistically significant(P<0.01). Compared with SHR group, the other group of the serum MDA level were significantly decreased(P<0.05). Compared with Nifedipine group, Compound QiMa capsule high-dose group of the serum MDA level were significantly decreased (P<0.05).(6) The comparison of serum SOD in each group. Compared with WKY group, SHR group, Nifedipine group, Nifedipine combine compound QiMa capsule and Compound QiMa capsule low-dose group of the serum MDA level were significantly decreased (P<0.05). Compared with SHR group, the other group of the serum SOD level were significantly increased (P<0.05). Compared with Nifedipine group, Compound QiMa capsule high-dose group of the serum MDA level were significantly increased (P<0.05).(7)The aortic morphology in WKY group that aortic intima was smooth, endothelial cells were intact, medial smooth muscles cells were in order. In SHR group, the aortic intimal continuity was destroyed, the endothelial cells local defected, in the irregular arrangement of smooth muscle cell. Aortic intimal in Nifedipine group was poor, medial smooth muscles cells were in order. Compared with nifedipine group, Nifedipine combine compound QiMa capsule and Compound QiMa capsule high-dose group of the arrangement of each layer cell endothelial cells did not see obvious defect or thickening.(8)Compared with the WKY group, SHR and Nifedipine group of eNOS mRNA expression were significantly decreased(P<0.05). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of eNOS mRNA expression were significantly raised (P<0.05). There were no significant among Nifedipine group, Nifedipine combine compound QiMa capsule group, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group(P>0.05).(9)Compared with the WKY group, SHR and Nifedipine group of Sirtl mRNA expression were significantly decreased (P<0.05). Compared with SHR group, Nifedipine combine compound QiMa capsule group, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of Sirtl mRNA expression were significantly raised (P<0.05). There were no significant among Nifedipine group, Nifedipine combine compound QiMa capsule group, Compound QiMa capsule high-dose group and Compound QiMa capsule low-dose group of Sirtl mRNA expression(P>0.05).(10) Compared with WKY group, SHR group and Nifedipine group of eNOS protein expression level were significantly decreased (P<0.05). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose and Compound QiMa capsule low-dose group of eNOS protein expression raised obviously (P<0.05). Compared with Nifedipine group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose and Compound QiMa capsule low-dose group of eNOS protein expression were significantly raised (P<0.05).(11)Compared with WKY group, SHR group NOX4 protein expression levels were significantly increased (P<0.05). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose and Compound QiMa capsule low-dose group of NOX4 protein expression level decreased significantly (P<0.05). Compared with nifedipine group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose and Compound QiMa capsule low-dose group of NOX4 protein expression level decreased significantly (P<0.05).(12)Compared with WKY group, SHR group of Sirtl protein expression level decreased significantly (P<0.01). Compared with SHR group, Nifedipine combine compound QiMa capsule, Compound QiMa capsule high-dose and Compound QiMa capsule low-dose group of Sirtl protein expression level raised obviously (P <0.05).Cloclusion(1)Endothelial cell injury in spontaneously hypertensive rat.(2)Compound QiMa capsule can increase the serum NO and reduce ET-1 and Ang II levels to improve endothelial cell function.(3)Compound QiMa capsule can increase the serum SOD and reduce the MDA level to improve oxidative stress of endothelial cells.(4)Compound QiMa capsule can protective the effect of SHR aortic endothelial structure.(5)Nifedipine combine compound QiMa capsule had advangage of better antihypertensive and protected vascular endothelium.(6)Compound QiMa capsule may promote the Sirtl and eNOS expression, and inhibit the expression of NOX4 in aortic endothelial cells to improve the level of NO, reduce oxidative stress to protect the vascular endothelium.Experiment Ⅱ Study on the protection and mechanism of Compound QiMa capsule medicated serum on HUVECs induced by Ang ⅡObjectiveTo investigate the Compound QiMa capsule medicated serum on the HUVECs and related factors induced by Ang II, and to research the protective mechanism of Compound QiMa capsule medicated serum.Methods(1)The activeness of cultivated HUVECs were assayed by MTT, which were incubated by the different concentrations’(10-8mol/L,10-7mol/L, 10-6mol/L,10-5mol/L,10-4mol/L) Ang II for different time(12h,24h,48h), and fininally we selected the Ang II was 10-6mol/L and the time is 24h to establish the model.(2)The activeness of cultivated HUVECs were assayed by MTT, which were incubated by the different concentrations’(5%,10%,20%) Compound QiMa capsule medicated serum for 12h,24h and 48h, and the result that the different concentrations’Compound QiMa capsule medicated serum no cytotoxicity.(3)HUVECs incubated for six group (①Blank group; ②Model group? ③5% Compound QiMa capsule medicated serum group;④10% Compound QiMa capsule medicated serum group;⑤20% Compound QiMa capsule medicated serum group;⑥atorvastatin group ),to collect HUVECs and culture medium after 24h for revelant test.(4)To observe the activeness of cultivated HUVECs induced by Ang II were assayed by MTT.(5)To absevev the morphological changes of HUVECs were observed under the light microscope.(6)The level of NO in each group excreted by HUVECs was essayed by nitrate reductive enzymatic test.(7)The level of ROS and apoptosis of HUVECs were essayed by flow cytometry technique.(8)The expression of eNOS, NOX4 and Sirtl mRNA in HUVECs were essayed by qRT-PCR.(9)The expression of eNOS, NOX4 and Sirtl protein in HUVECs were essayed by Western Blot.Results(1)Compared with the blank group, the activeness of the model group was significant decreased(P<0.01). Compared with the model group, the activeness of the other groups were significant increased(P<0.01).(2)The cells morphological were observed after 24 hours under inverted microscope. Blank group of HUVECs showed flat circular fusiform paving stone pattern, obvious nucleolus, outline clearly. The model group, most cells shrivel assumes the circular or elliptic, intercellular gap get bigger, unclear nucleoli. And the HUVECs which intervened by compound QiMa capsule medicated serum showed that some cells shrivel assumes the circular or elliptic, intercellular contact more closely and nucleolus is clear, and 10% compound QiMa capsule medicated serum group was better than that lower concentration of serum medicated. After atorvastatin treatment of cell morphology is similar to the normal group.(3) Compared with the blank group, the model group of NO level was decreased obviously, the difference was statistically significant (P<0.01). Compared with model group,10% compound QiMa capsule medicated serum group and atorvastatin group of NO level were increased(P<0.05). There were no significant difference among model group,5% compound QiMa capsule medicated serum group and 20%compound QiMa capsule medicated serum group(P>0.05).(4) Compared with the blank group, the other groups of HUVECs level of ROS were obviously increased (P<0.01). Compared with model group, the other groups of HUVECs level of ROS were also obviously increased(P<0.01). ROS levels between the drug intervention groups were significant differences, of which 10% compound QiMa capsule medicated serum group of intracellular ROS content is the lowest, followed by atorvastatin group, again is 20% compound QiMa capsule medicated serum group, ROS content was highest in 5% compound QiMa capsule medicated serum group (P<0.05).(5)Compared with the blank group, the other groups of HUVECs level of apoptosis rate were obviously increased(P<0.01). Compared with model group, the other groups of HUVECs level of apoptosis rate were also obviously increased (P<0.01). Apoptosis rate levels between the drug intervention groups were significant differences, of which 10% compound QiMa capsule medicated serum group of apoptosis rate is the lowest, followed by atorvastatin group, again is 20% compound QiMa capsule medicated serum group, apoptosis rate was highest in 5% compound QiMa capsule medicated serum group (P<0.05).(6)Compared with the blank group, each group cell eNOS mRNA expression decreased obviously, the difference was statistically significant (P<0.05). Compared with model group,10% compound QiMa medicated serum group and atorvastatin group eNOS mRNA expression level increased obviously, the difference was statistically significant (P<0.01). Compared with 10% compound QiMa medicated serum group and atorvastatin group, apocynin group and sirtinol group eNOS mRNA expression were decreased obviously, the difference was statistically significant (P<0.05). There were no statistical significance between Apocynin group and sirtinol grouop (P>0.05).(7)Compared with the blank group, the other group of NOX4 mRNA expression increased obviously, the difference was statistically significant (P<0.01). Compared with model group,10% compound QiMa medicated serum group, atorvastatin group and apocynin group NOX4 mRNA expression level decreased obviously, sirtinol group increased obviously, the difference was statistically significant (P<0.01). Compared with sirtinol group,10% compound QiMa medicated serum group, atorvastatin group and apocynin group NOX4 mRNA expression were decreased obviously, the difference was statistically significant (P<0.01).(8) Compared with the blank group, model group, apocynin group and sirtinol group Sirtl mRNA expression decreased obviously, the difference was statistically significant (P<0.05). Compared with model group,10% compound QiMa medicated serum group and atorvastatin group Sirtl mRNA expression level increased obviously, sirtinol group decreased obviously, the difference was statistically significant (P<0.01). Compared with 10% compound QiMa medicated serum group and atorvastatin group, apocynin group and sirtinol group Sirtl mRNA expression were decreased obviously, the difference was statistically significant (P<0.05). Compared with apocynin group, sirtinol group Sirtl mRNA expression were decreased obviously(P<0.05).(9)Compared with the blank group, the other group of eNOS protein expression decreased obviously, the difference was statistically significant (P<0.01). Compared with model group,10% compound QiMa medicated serum group and atorvastatin group eNOS protein expression level increased obviously, the difference was statistically significant (P<0.01). Compared with 10% compound QiMa medicated serum group and atorvastatin group, apocynin group and sirtinol group eNOS protein expression were decreased obviously, the difference was statistically significant (P<0.05). Compared with the sirtinol group, apocynin group eNOS protein expression was increased. (P<0.01).(10)Compared with the blank group, the model group,10% compound QiMa medicated serum group, atorvastatin group and sirtinol group NOX4 protein expression increased obviously, the difference was statistically significant (P<0.01). Compared with model group,10% compound QiMa medicated serum group, atorvastatin group and apocynin group NOX4 protein expression level decreased obviously, sirtinol group increased obviously, the difference was statistically significant (P<0.01). Compared with 10% compound QiMa medicated serum group and atorvastatin group, sirtinol group NOX4 protein expression were increased obviously, the difference was statistically significant (P <0.01). Compared with sirtinol group, apocynin group NOX4 protein expression were decreased obviously(P<0.01).(11)Compared with the blank group, the other group of Sirtl protein expression decreased obviously, the difference was statistically significant (P<0.05). Compared with model group,10% compound QiMa medicated serum group and atorvastatin group Sirtl protein expression level increased obviously, the difference was statistically significant (P<0.01). Compared with 10% compound QiMa medicated serum group and atorvastatin group, apocynin group and sirtinol group Sirtl protein expression were decreased obviously, the difference was statistically significant (P<0.05). Compared with sirtinol group, apocynin group Sirtl protein expression were significant increased (P <0.05).Conclusion(1)The concentration of 10-6 mol/L AngⅡ intervention 24 h can be used for production of HUVECs damage model.(2)10% compound QiMa medicated serum can alleviate the morphological of HUVECs induced by AngⅡ.(3) 10% compound QiMa medicated serum can improve AngⅡ cultivating HUVECs NO levels in the model, and inhibit the production of ROS, alleviate the damage caused by oxidative stress to cells.(4) 10% compound QiMa medicated serum can reduce apoptosis which induced by Ang Ⅱ.(5) eNOS and Sirtlexpression can be inhibited by AngⅡ cultivating HUVECs and promote expression of NOX4, and cause endothelial cell injury.(6)compound QiMa medicated serum and atorvastatin can improve eNOS and Sirtlexpression and inhibit expression of NOX4 in AngⅡ cultivating HUVECs and play a role of protection of endothelial cells.(7)Compound QiMa medicated serum may promote upstream Sirtl gene expression, then increase eNOS expression to promote the generation of NO, decrease NOX4 expression to reduce the production of ROS to protect endothelial cell function.