| Artherosclerosis(AS) is one kind of disease which seriously harms the people' health and involves the important organ, such as heart, brain, to cause severely consequence. As the life level increasing and the development of social economy ,the incidence of AS appears the rising trend. So the medical science have paid generally attention to t the anti-AS study .The dissertation which includes three aspects , i.e. literature study, experiment study and clinical research respectively, investigated the correlative nosogenesis, the anti-AS action mechanism of ZHIMAINING capsule by using cell culture ,animal experiment, clinical observation.Part one : literature studyThe abnormality of lipid metabolism , hypertension,. smoking, diabetes mellitus, adipositas, heredity can affect the morbility of AS and are the accepted dangerous factors. Some new dangerous factors are found and confirmed in recent study, for example homohalfcystineemia, lipoprotein, triacylglycerol, disfunction of blood coagulation and lysodelicate system, infection, oxidative stress, inflammatory reaction, et al. The others factors, such as the cell proliferation and apoptosis.the AS-related gene (LDL-R,Ox-LDL,VCAM, Angll), endothelin, the abnormal expression of NO, all have the direct relationship with the occurrence and succession of AS.The traditional Chinese medical science thinks thatAS belongs to the category of expectoration symptom and vascular arthralgia. AS was generally induced byimproper diet> function disturbanceof entrain inherent factor which expectoration was produced by body and block the blood circulation. The expectoration % congestion or the interaction of expectoration and congestion should be regarded as the main symptom in differentiation of symptoms and signs. The therapy abide by the following principle, such as eliminating phlegm-, strengthening body resistances regulating vital energy > clearing the pyretic toxicity, et al.Part two: experiment study2.1 The ZHIMAININGH protective action on VEC-304 cell damage induced by cigarette extract(CSE)Aim: Investigating the influence of ZHIMAINING drug serum on VEC-304 cell survival ratio and apoptosis percentages cell apoptosis protein expression> oxidation stress reaction in cell lysate by using blood serum pharmacology method.Method: The blank serum and drug serum was prepared respectively by using SD rats. The VEC-304 cell was divided into 4 groups: ?Control group: The blank serum was added into cells;?CSE group: The cigarette extract(CSE) and blank serum was added into cells respectively;?Drug group: The drug serum was added into CSE group;?Drug group II: The same proportion of blank and drug serum was added into CSE group. The cell lysate was collected and the content of SOD, GSH-Px, GSH, MDA was determined;After the cell DNA was extracted and cell section was prepared, cell apoptosis percentage and survival ratio was assayed by using TUNEL and Ladder electrophoresis;The protein expression of Bcl-2% Bax> Catapase-3 was determined by using Western blot and cell immuno -histochemistry;The survival ratio was determined by using MTT method.Results: The survival ratio and apoptosis percentage of control group was 91.3% and 11.2% respectively. CSE-induced group was 61.6%, 34.7% respectively. There was significant difference comparing the control group with CSE-induced group. The ZHIMAINING drug serum-treated group I was 84.5% and 17.3% respectively. The ZHIMAINING-treated group II was 69.5% and 19.5% respectively. The cell survival ratio of drug serum group was significant higher than CSE-induced group, but the apoptosis percentage was lower (p<0.05-0.01). In the dose range, there lied the dose -effect relationship.The expression of P2K Caspase-3-. Bax in CSE-induced group was obviously higher than control group except for Rb, but the Bcl-2% MP53 level obviously decreased(p<0.05-0.01). The expression of RK P2K Caspase-3 in drug group all down regulated. Bcl-2, MP53 level up regulated. Comparing with CSE-inducedgroup ,the P value was lesser than 0.05-0.01.The activity of SOD, GSH-Px, T-AOC, GSH in VEC-304 cell induced by CSE all decreased, but the MDA content and the NOS activity increased. There was significant difference comparing with control group except for SOD activity. The activity of GSH-px, T-AOC, GSH was obviously higher than CSE group (P<0.05-0.01),but the NOS activity decreased(P<0.05).2. 2 The influence of ZHIMAINING on theVEC-304 cell apoptosis and oxidation impairment induced byH2O2.Method: The VEC-304 cell was divided into 4 groups. (DControl group: The blank serum was added into cell;?H2O2 group: The 0.5mmol/L H2O2 and blank serum was added into cells respectively;?Drug group I :The durg serum was added into the H2O2 group;@Drug group II: The same proportion of blank and drug serum was added into the H2O2 group. The cell was cultured for 48h,then the clear supernatant was discarded. Cell of all group was collected and made into cell section. After the cell lysate was collected and cell DNA was extracted, the cell survival ratio % cell apoptosis percentage > apoptosis protein expression and the related oxidation index was determined rexpectively by using immunohistochemistry, TUNEL and Ladder method.Results: The survival ratio of H2O2-induced VEC-304 group was lowest and the percentage of apoptosis was highest. The P value was lesser than 0.05. ZHIMAINING can obviously raised the survival ratio and deduced the apoptosis percentage of H2O2-induced cell. There was significant difference between drug groups (PO.05).The expression of Caspase-3, Bax of drug group cell was obviously higher than the control group(P<0.01), but the Bcl-2 value decreased obviously(P<0.01). The drug group can down-regulated the expression of Caspase-3 , Bax> and up-regulated Bcl-2 expression .When comparing the drug and H2O2 group, The difference was significant (PO.01). But the difference of Bcl-2, Bax, Caspase-3 % NF-Kb expression of drug group I and II was also significant (PO.05-0.01).The activity of SOD, GSH-Px> T-AOC and content of GSH all decreased in H2O2-induced VEC-304 cell, but the MDA content increased.The difference of above-mentioned indexes was significant except for the SOD activity(P<0.05-0.01). But the indexes of drug group was adverse to the H2O2 group. There was significant difference between drug groups (PO.05-0.01 ).The results showed that ZHIMAINING had significant antioxidation activity which can inhibit the cellmembrane peroxidation damage induced by IkC^.The efficacy of inhibit the cell membrane peroxidation damage was related with the drug dose.2.3 Influence of ZHIMAINING on NF-kB^ HSP-70 expression of VEC-304 cell induced by CSE and H2O2Methods: Investigating the NF-kB > HSP-70 expression of VEC-304 cell induced by CSE and H2O2 and intervention action of ZHIMAINING SD rat serum by using immunohistochemistry-. TUNEL and Ladder method.Results: The NF-kB > HSP-70 expression in CSE group all were stronger than control group. The results indicated that the expression of NF-kB > HSP-70 all were activated and obviously up-regulated with the CSE stimulus. The NF-k^ HSP-70 expression of VEC-304 cells induced by CSE in experiment I and II group with the treatment of ZHIMAINING serumdown-regulated (PO.05~0.01, which showed that ZHIMAINING can protect the cell damage caused by CSE. There were significant difference of down-regulation of NF-kB between experiment I and II group (PO.05), which indicated there was dose dependent relationship. The Western blot analytic results showed that positive strap and P-actin ratio increased and the P value was lesser than 0.01 comparing the CSE group and control group. The positive strap of HSP-70 > NF-kB and P-actin ratio in experiment I and II group decreased comparing with CSE group (P<0.01).The expression of NF-kB -. HSP-70 in H2O2 group was obviously stronger than control group(PO.05~0.01. The NF-kB > HSP-70 expression of VEC-304 cell with the treatment of ZHIMAINING serum in experiment I and group significantly down-regulated comparing with the H2O2 group (PO.05~0.01 ) . There was significant difference (PO.05) between experiment I and II group, which indicated that ZHIMAINING can protect the cell damage induced by H2O2. The Western blot analytic results showed that the positive strap of HSP-70 ^ NF-kB and P-actin ratio increased comparing the control group and H2O2 group (PO.01) .2.4 Influence of MAIZHINING capsule on AS rabbit oxidation stress and gene expression of LDL-R, VCAM-1Methods: Rabbit AS model was copied by using hypsilipid forage. The hyperlipoidemia appeared in the 4th week, and then the rabbits were given WEIMAINENG capsule by intragastric administration for 8 weeks. The experiment period was total 12 weeks. After the experiment completed .rabbits blood was drawn out and serum was separated. The arteriae aorta LDL-RmRNA% VCAM-lmRNA expression level was determined by using tissue in situ hybridization and immuno-histochemistry method and compared the pathological changeResults: Serum TC> TG> HDL^ LDL level of AS rabbit induced by high cholesterol forage all increased and were obviously higher than blank group (P<0.01). The above- mentioned indexes of drug group were also higher than blank group, but obviously lower than model group (PO.01). There were significant difference of TC\ TG% LDL content between drug groups.The SOD-* MDA> T-AOC level of model group were all distinguished higher than blankgroup(P<0.01)and GSH .. GSH-Px level decreased(P<0.01).The above-mentioned indexes of drug group I also obviously increased (PO.05-0.01), but significantly lower than model group, especially lipid peroxidation MDA content (PO.05). The difference of SODn MDAn GSH-px activity of drug group II was significant comparing with the model group (PO.05-0.01). The antioxidase SOD activity and total antioxygen T-AOC level of model increased. It was the compensative increasing of AS rabbit under high oxidation stress state to cause this phenomenon and was one kind of self-adjustment mechanism. The results indicated that ZHIMAINING capsule had significant antioxidation action.The arteriae aorta LDL-RmRNA expression of model rabbit was obviouslyinferior than blank group (PO.01), but the VCAM-lmRNA expression was stronger(P<0.01).The LDL-RmRNA expression of drug group was stronger than model group(P<0.01) , but VCAM-lmRNA expression was inferior (PO.01. Drug group I wasmore significant than drug group II (PO.05) .The blank group rabbit arteriae aorta microstructure and the tunica media elastic fibers were normal, and the endomembrane was intact. There were obvious accrementition eminentia and a lot of smooth muscle cell proliferation and bulky foam cells in model group. The accrementition of arteriae aorta endomembrane was not apparente. The smooth muscle cell and elastic fibe proliferation was not clear in drug group I .There were longitudinal local arteriae aorta endomembrane proliferation % sparing foam celK smooth muscle cell and connective tissue in drug group II.Part three: Clinical study3.1 Influence of MAIZHINEVG capsule on blood pressure of CAS patient complicated with hypertensionMethods: 33 AS patient complicated with hypertension were randomly divided into 2 groups. One group took ZHIMAINING capsule;the other 17 patient took XUEZHIKANG as control group.CDAfter the single dose administration,continuously observed the change of blood pressure in 3h;?The hypotensive drug dose changed from the beginning of course of treatment to the 4th week.Results: The acute depressurization experiment indicated that blood pressure descended gradually after taking ZHIMAINING capsule orally in 0.5h and about 2-3h later drug action approached the maximum. The drug action difference of lh â– ? 2h -. 3h was significant than pretherapy (PO.01). 3h after taking the XUEZHIKANG orally patient blood pressure of control group kept stable and the change was not clear. The drug action of the ZHIMAINING group was all significant higher than the control group in lh> 2h> 3h respectively. Long-term therapy results indicated that after 2 week therapy using ZHIMAINING capsule, the western medicine all can be stopped or reduced. After 4 week therapy 37.5 percentage of patient can stop using the western medicine. Nearly 62.5 percentage of patient can reduce 50% drug dose . But 4 weeks later there was no patient who stopped using the western medicine in XUEZHIKANG therapy group. Only 29.4 percentage of patient can reduce 50% drug dose.The statistical results indicates that the antihypertension action of ZHMAI -NING capsule was more significant than XUEZHIKANG3.2 Influence of ZHIMAINING capsule on LDL-R> VCAM-1 gene expression and oxidation stress of carotid artery scleratheroma (CAS) patientMethods: The 72 CAS patient were randomly divided to 2 group: The patient number of therapy group was 36, who took ZHIMAINING capsule orally as drug group;The remaining patient as control group took XUEZHIKANG Blood fat and oxidizing reaction were selected as the compared index with 20 normal people. The peripheral blood lymphocyte LDL-RmRNA> VCAM-lmRNA expression level was determined by using in situ hybridization. Carotid artery endomembrane- tunica media thickness (IMT) was determined by using ultrasonic scanning.ResuIts:The value of IMT was obviously lesser than pretherapy(P<0.05). The reduction degree of drug group was bigger than control group, but statistical significance was not obvious.TC> TG> LDL level > atherosclerosis index (AT) and IMT all obviously descended in therapy group. The HDL value increased obviously. There were significant difference comparing with pretherapy (PO.01), also were the control group except for HDL (PO.05-0.01). But the curative effect of therapy group was better than control group which indicates that the regulating lipid metabolism action of ZHIMAINING capsule surpassed the XUEZHIKANGLDL-RmRNA expression all was obvious lower than normal value (PO.Ol)and VCAM-lmRNA expression was higher before therapy (PO.01). After therapy LDL-RmRNA expression was up-regulated and VCAM-lmRNA expression was down-regulated in therapy group(P<0.01), so as the control group(P<0.05). There was significant difference between pretherapy and post-treatment (PO.01), so as the therapy and control group (p<0.05).SOD activity all was lower than normal people and LPO content was higher in both groups (PO.01). After therapy SOD activity rose (PO.01) and LPO content descended (PO.05-0.01). The difference of two groups was significant (PO.05). But the therapy group curative effect outweighed the contro 1 group.4 ConclusionsZHIMAINING capsule had protection action for VEC-304 cell apoptosis and oxidation damage induced by CSE and H2O2. Regulating the related cell apoptosis gene expression^ blocking the NF-kB signal conduction path> increasing the Bcl-2/Bax ratio > inhibiting the Caspase cascade reaction and cell apoptosis was the possible action mechanism.ZHIMAINING capsule can significantly lessen AS rabbit arteriae aorta plaque by regulating the lipid metabolism > inhibiting the oxidation reaction ^ regulating LDL-R, VCAM-1 gene expression.ZHIMAINING capsule can regulate the lipid metabolism n inhibit the oxidation reaction regulate LDL^ VCAM-1 gene expression to lessen the arteriae aorta endomembrane- tunica media thickness(IMT) of CAS patient.ZHIMAINING capsule produce the therapeutic effect 0.5h after taking the capsule orally by using acute depressurization experiment. 2-3h later the therapeutic effect approached the maximum. It can reduce the dose or stop using the western medicine quickly by long-term continuous applying. The curative effect is better than XUEZHIKANGZHIMAINING capsule can regulate the lipid metabolism > inhibit cell apoptosis % antioxidatio^ regulation related gene expression. So it can lessen and eliminate the artherosclerosis plaque. The action characteristic of ZHIMAINING capsule showed dose-effect relationship. |
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