| The material foundation of efficacy of Traditional Chinese Medicine (TCM)is the hot and difficult spot of modern research of the theory of compound compatibility, development of new drug and clarifying the modern connotations for syndrome differentiation The biological effects of Chinese medicinal formulae and the effectiveness of syndrome differentiation and treatment all can hardly be revealed without the guidance and confirmation of medicinal material base Therefore, revealing the the material foundation of efficacy of Chinese herbs is always key issue for modernization of TCM. However, due to the complexity of the chemical constituents of Chinese herbs and the use of compound preparation, the interactions between each element could be extremely complex and the identification of active compounds could be rather difficult. Even if the Chinese herbs documented in Chinese Pharmacopeia(2010edition), the varieties of traditional Chinese medicine with definite functional components makes up less than5%。At the present, the index components of the most Chinese herbs for quality control were set depended on the content of the components. So they were not necessarily the the main efficacy components。This problem is not only restricting the modernization of TCM from achieving substantial progress, it also can easily lead to drug safety. Therefore, the research of material foundation of efficacy of TCM is also the difficult and crucial part in modernization of TCM, which should be solved urgently.The team of my tutor have striven to the research of material foundation of efficacy of TCM for years, To deal with this problem, my tutor had put forward the idea of utilizing method of "knockouts" basing on the monoclonal antibody of small molecules of TCM, which differed from the method of reductionism. Through knocking out some component of a TCM specifically, we can compare the differences and similarities of efficacy before and after knocked out and the single component. The method could not only reflect the effect of the single compoent isolated from the mix, but also could show the role the component played within the herb. Therefore, the possible connections between regulators and other circuit components and the true contribution to efficacy of herb could be revealed-PRR (PRR) is the documented index components of PRA (PRA) for quality control. PRA and PRR have definite effect in the treatment of ischemic stroke and the mechanism is relatively clear.On the basis of many years of research, although lots of mechanism of PRR for ischemic stroke have been set out, the contribution of PRR to PRA has not been reported。 So we chose PRR as pattern drug, the vitro mode of ischemic stroke—oxygen glucose deprivation (OGD)in SH-SY5Y cells as carrier, intended to reveal the contribution of PRR to PRA in regulation of the Inflammatory factors in the model by comparing the function of PRA before and after the PRR was knocked out specially.Chapter1Preparation of immunoaffinity column and the knocking out of PRR in PRA exactObjective:To Prepare immunoaffinity column basing of the PRR monoclonal antibody. Then the PRR in PR exact should be knocked out and the drug for follow-up study should be prepared.Method:The ascites containing PRR monoclonal antibody(Mab) was purified by method of octanoic acid-ammonium sulfate; purity of antibodys before and after were tested by SDS-PAGE; concentrations of antibodys were measured by Bradford quantitative determination;the titer and competition of antibodys were detected by ELISA; the purified PRR Mab was coupled with the CNBr-activated SepharoseTM4B and the conjugation efficiency and conjugation rate were computed; PRA-exact(PRA-EXT) was loaded in the immunoaffinity column and the concentrations of PRR in PRA-EXT and the knock out couple (PRA-KO)were tested by HPLC, the rate of knock out was calculated.Result:SDS-PAGE showed that the other protein in PRR Mab were reduced, the purity of PRR Mab was92%; the concentration of PRR Mab ascites is3.67mg/mL, the concentration of the purified PRR Mab is2.29mg/mL; the ELISA showed that the titer of PRR Mab are both1:51200before and after purified, no apparent changes of linearity rangewere observed; the conjugation efficiency is99.3%, conjugation rate is3.1mg/mL; the90%of PRR was knocked out form PRA-EXT.Conclution:The PRR Mab purified by by method of octanoic acid-ammonium sulfate with less Miscellaneous protein and without influence to the titer and competition, met the requirement for preparation of immunoaffinity column, the conjugation between PRR Mab and the CNBr-activated SepharoseTM4B was well and the rate of knock out could meet the demands of follow-up study.Chapter2Comparative study on the pharmacodynamics of PRA-EXT, PRA-KO and PRR on the OGD injury of SH-SY5Y cellsObjective:Studying the pharmacokinetics of PRR in SH-SY5Y cells to reveal the efficacy generation process. To establish a stable oxygen glucose deprivation-reperfusion (OGD) in SH-SY5Y cells. To compare the differences between pharmacological activities of PRA-EXT, PRA-KO and PRR for this model.Method:(1)The effect of PRR on normal SH-SY5Y cells was studied with8different concentrations(3.9,7.8,15.6,31.3,62.5,125,250,500μg/mL) and4different incubation time(2h,8h,16h,24h); Cells were incubated with3different concentrations of PRR (31.25μg/mL,62.5μg/mL,125μg/mL) respectively, the supernatant was collected at20timepoints sequentially (0.08,0.25,0.5,0.75,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,8,9,10h) and tested by icELISA for the concentration of PRR. The intracellular concentration of PRR was obtained by subtracting the concentration of PRR in supernatant from the total dosage.(2) The OGD model in SH-SY5Y cells were reduced by sugar-free cell-culture medium and Na2S2O4, the condithons of Na2S2O4with4different concentrations (0.4,0.8,1.6,3.2mmol/L) and4different incubation time (2,8,16,24h) was investigated. Cytomorphology was observed under optic microscope; Cell survival rate was tested by MTT assay, Activities of lactate dehydrogenase (LDH) also be detected.(3) The PRA-EXT, PRA-KO and PRR was used to intervene the model with nine different concentrations of PRR (3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL) individually. Cell survival rate was tested by MTT assay, Activities of lactate dehydrogenase (LDH) also be detected.Results:(1)PRR of3.9,7.8,15.6,31.3,62.5,125,250μg/mL had no significant effect on cell viability and LDH levels within8hours; The AUCiast and Cmax of high, medium and low dose groups decreased in a dose-dependent manner, The Tmax and MRTiast of medium dose group was higher than that of low-dose group and high-dose group, which showed the he particularity of the medium dose absorbed process in the cell.(2)The result of MTT and LDH test showed that the cell survival rates of cells in groups with dose of Na2S2O4is0.8,1.6and3.2mmol/L were decreased remarkablythan that in the control groups(P<0.01); the level of LDH in supernatant of cells in groups of0.4,0.8,1.6and3.2mmol/L were significantly higher than that in the control groups.(3)The result of MTT and LDH test showed that the cell survival rates and the level of LDH in supernatant of cells in PRR groups with dose of PRR is15.6,31.3,62.5,125and200μg/mL were improved remarkably(P<0.01or P<0.05) than that in the model groups; which means the damage caused by the model can be inhibited by PRR, indicating successful model. The best dose of PRR is62.5μg/mL, which may be related to the specific intracellular absorption process..The cell survival rates and the level of LDH in supernatant of cells in PRA-KO groups with dose of PRR(Before kocked out) is15.6,31.3,62.5,12,200,250,500μg/mL were improved remarkably (P<0.01or P<0.05) than that in the model groups; The cell survival rates and the level of LDH in supernatant of cells in PRA-EXT groups with dose ofPRR is3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL were improved remarkably(P<0.01) than that in the model groups; The best dose was250μg/mL. Within15.6-250μg/mL range the efficacy of PRA-EXT groups was better than that of PRA-KO groups.Conclusion:Taken within the dose range of this experiment, the effect of PRA-EXT was better than the effect of PRR showed the combined effects of a variety of ingredients was better than single ingredient used alone; PRA-EXT performed better than PRA-KO indicated that knockout90%PRR from EXT will weaken the overall efficacy, PRR had a relationship with the overall efficacy of PRA-EXT; the effect of PRA-EXT was better than that of both PRA-KO and PRR used alone illustrated PRR may have a synergistic effect with other components of Pueraria.Chapter3Research on the dose-effect relationship between PRR and the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injuryObjective:To reveal the dose-effect relationship between PRR and the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury by the comparison amang the PRA-EXT, PRA-KO and PRR on each optimal dose, as well as the comparison amang the PRA ontaining different proportions of PRR.Method:(1)According to the best dosage of the three drugs elected in the Chapter l,the OGD model of SH-SY5Y cells were treated by the PRA-EXT, PRA-KO and PRR on3different concentration of PRR for comparing the efficacy of them;(2)At the best dose of PRA-EXT and the corresponding dose of PRA-KO, PRR was quantitatively added into PRA-KO (the content of PRR is10%) to the content of25%for the PRA-EXT, then comparing the efficacy of PRA-KO(the content of PRR is10%), PRA-KO (the content of PRR is25%) and PRA-EXT (the content of PRR is100%);Tested the cell survival rate and LDH level; SOD activities were measured by xanthine oxidase method; the content of MDA were measured by thiobarbituric acid method;the content of NO was detected with Griess method;the expression of NF-κB、TNF-αand IL-6were was investigated by Western blot.Result:(1)At the best dose of PRR, the improving effect of PRA-KO on abnormal change of cell survival rate,LDH level, NF-κB, TNF-α, SOD, MDA, NO caused by OGD were worse than these of PRA-EXT, the improving effect of PRA-KO on IL-6is better than PRR, but non difference with PRA-EXT; at the best doses of PRA-EXT and PRA-KO, the improving effect of PRR on cell survival rate,LDH level, NF-κB, TNF-α, SOD, MDA, NO, IL-6were worse than these of PRA-EXT and PRA-KO; the improving effect of PRA-KO on cell survival rate,LDH level, NF-Kb, TNF-α SOD, MDA, NO were worse than these of PRA-EXT;(2)The improving effect of PRA-KO(the content of PRR is25%) on cell survival rate, LDH level, NF-κB, TNF-α, SOD, MDA and NO were better than these of PRA-KO(the content of PRR is10%) and PRR (which equal to the dose added in PRA-KO), but still worse than PRA-EXT (the content of PRR is100%).Conclusion:(1) In three different concentrations of PRR selected in the present study, the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury was weaken by knocking out90%PRR from PRA, indicating there was a relationship between the efficacy of PRA and PRR; the effect of PRR on the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury included reducing NF-κB, TNF-α levels, improving SOD levels, reducing MDA, NO, but the effect on IL-6is relatively weak. This experiment provided the direct evidence for the relationship between PRA and PRR.(2) After PRR was quantitatively added into PRA-KO (the content of PRR is10%) to the content of25%, the improving effect of PRA-KO(the content of PRR is25%) was enhanced and better than PRR and PRA-KO use alone, which means synergies existed between PRR and the components of PRA-KO; The improving effect of PRA-EXT (the content of PRR is100%) is better than that of PRA-KO (the content of PRR is25%) and PRA-KO(the content of PRR is10%), indicating the influence of PRR on the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury worked in a dose-dependent manner; This effect mainly related to the regulation of NF-κB, TNF-α. SOD, MDA and NO. |
PDF Full Text Request