| ObjectiveTo investigate the intervening effect of composed of formula based on the basic therapeutic principles of Qi-Boosting Toxin-Resolving on cytoskeletal protein and its implications in the potentiality of motility of nasopharyngeal carcinoma cell line CNE2Z to explore the potential significance and possible clinical usage of these basic mechanisms.Experimental ProceduresPart One. The main formulae composed based on the therapeutic principles of Qi-boosting Toxin-resolving and their basic pharmacological characteristicsMethods Two herbal medicinal formulae, the original formula and revised one, were composed based on the therapeutic principles of Qi-Boosting Toxin-Resolving at first. Then, medicinal blood plasma was prepared in a routine way for these two formulae among rats, with another group of animals was taken as healthy blank control. Followed were the experimental steps taken to determine the non-toxic and toxic concentrations of these plasma, based on the outcomes of proliferating activity-inhibiting assay and determination on their IC50 on the target cells of nasopharyngeal carcinoma cell line CNE2Z to explore their basic pharmacological characteristics.Results There was no obvious toxic effect on the target cells for healthy rat plasma when it was used at a concentration equal to, or lower than, that of 15%. It was deducted with the IC50 concentration of the medicinal blood plasma for original formula group being 8.4% based on a fitting progress with a regression equation. However, there was no significant inhibiting effect on the proliferating activity of target cells for the medicinal blood plasma of revised formula, so the used plasma concentrations in the followed experiments were the same as that of original one. The highest non-toxic concentrations were all 2.50% for both of groups of original and revised formulae plasma. However, the medicinal blood plasma of original group showed mild level of promoting effect on the target cells at the concentrations of 1.25%. Therefore, two kinds of non-toxic concentrations of 2.50% and 1.25% were used in the following experimental steps for each formula respectively.Part Two. The effects of different kinds of Qi-Boosting Toxin-Resolving formulae on expressing activity of main responsible genes for the potential motility of CNE2Z cells Methods Two different non-toxic concentrations of the medicinal blood plasma from two formulae were taken to treat cells CNE2Z respectively, with the blank blood plasma as the control, to detect the changes in the expressing activities of mRNA of the main responsible genes nm23 and c-myc with the procedures of RT-PCR and the expressing levels of protein of these two indicators by immunocellularchemistry SABC, followed by comparative and correlative analysis on these results to find out the intervening mechanisms, their possible biologic significance and the potential clinical significance in the future.Results Based on the outcomes of immunocellularchemistry SABC, the result showed that there was no significant difference in the expressing level of c-myc among the tested groups, while that of nm23-H1 was shown with statistical significance (P＜0.01) for the experimental results of original formula, suggesting that original formula holding the effect to promote expressing activity of nm23-H1 in CNE2Z. However, it was shown that revised formula could promote the expressive level of c-myc but to inhibit the expression of nm23-H1 in the target cells, with statistical significance (P＜0.01).As shown from the experiments of RT-PCR, the expressive levels of nm23-H1 mRNA were different among various dosages for both of original and revised formulae, with significant differences for high and medium dosages but not for low dosage as shown by OD values of these tested gels.Part Three The effects of different kinds of Qi-Boosting Toxin-Resolving formulae on the expressing activity of main responsible cytoskeletal proteins for the potential motility of CNE2Z cellsMethods Two different non-toxic concentrations of the medicinal blood plasma from two formulae were taken to treat cells CNE2Z respectively, with the blank blood plasma as the control, to detect the changes in the expressing activities of ytoskeletal proteinsβ-tublin, F-actin, ezrin with SABC procedures, and to detect the changes in the transcriptional level of ezrin mRNA with RT-PCR in order to investigate the intervening mechanisms, their biologic significance and the potential clinical implications in the future.Results As shown by the SABC procedures, the original formula could inhibit the expression ofβ-tubulin, with very high statistical significance (P<0.01), while the revised formula could promote the expression of this protein in CNE2Z at high dosage, also with very higher statistical significance (P<0.01), but the medium and lower dosages showed inhibitory effect on the expressive levels ofβ-tubulin in the target cells with statistical significance (P<0.01).The expressive level of F-actin could be inhibited significantly in CNE2Z by original formula (P＜0.01), while its expression could be promoted significantly by revised formula (P<0.01).The expressive activity of p-Ezrin was shown significantly inhibited by original formula (P＜0.01), while its expression was obviously promoted by revised formula at higher dosage in CNE2Z (P<0.01), though its expression was inhibited at medium and lower dosage (P＜0 .01).Part Four The effects of different kinds of Qi-Boosting Toxin-Resolving formulae on the potential motility of cells CNE2Z and the correlative analysis on the association of potential motility with the expressive activities of their main responsible genesMethods Two different non-toxic concentrations of the medicinal blood plasma from two formulae were taken to treat cells CNE2Z respectively, with the blank blood plasma as the control, to detect the changes in the potential motility of cells CNE2Z by scratch test, followed by observation on the morphologic characteristics and pseudopod of these cells by both of electron microscopy and light microscopy to determine their changing features in potential motility in the responsibility to the intervening plasma in order to explore the intervening mechanisms, biologic significance and the potential clinical simplications in the future.Results When cultured with the medicinal blood plasma of original formula at higher dosage for 24 hrs, the changes in potential motility were shown very significant (P<0.01), but not at its lower concentration.Electron microscopy revealed that the target cells showed looser growing status in the original group than that in control one, with the pseudopod in the original group looking like silkiness and expansion not as obvious as in control group. The cells were grown more intensively than in control group when intervened by revised formula, with longer pseudopod and more obvious expansion as in controlling group.Part Five. The intervening mechanism of different kinds of Qi-Boosting Toxin-Resolving formulae with the signal transduction network underlying the potential motility of cells CNE2ZMethods Two different non-toxic concentrations of the medicinal blood plasma from two formulae were taken to treat cells CNE2Z respectively, with the blank blood plasma as the control, to detect the changes in the expressive activities of p-JNK and Rac 1 before and after intervening in the target cells with SABC procedures to explore the possible mechanisms responsible for signaling network underlying the potential motility, the biologic significance and the potential clinical implications in the future.Results Original formula could obviously inhibit the expression of Rac-1 (P<0.01), while the revised formula could significantly promote the expression of (3-tubulin in CNE2Z at high dosage (P<0.01), though its medium and lower dosages did not showing such an inhibitory effect on the expression of this protein.The origin formula could also significantly inhibit the expression of JNK in CNE2Z, with the result showing obvious statistical significance (P＜0.01).Conclusions1. The original formula composed of on the same therapeutic principles of Qi-boosting toxin-resolving can significantly affect the process of pseudopod formation and remarkably inhibit cellular potential motility of NPC cell line CNE2Z. In the contrast, the revised formula can promote the process of pseudopod formation at some levels and increase its potential motility.2. The origin formula composed of on the same therapeutic principles of Qi-boosting toxin-resolving can significantly promote the expressive activity of nm23-H1, the main responsible gene for potential motility, while the revised formula can obviously inhibit its expressive level in CNE2Z. These results indicate that different formula composing methods of herbal medicines, even based on a same therapeutic principle for a same condition, can induce significantly different pharmacological effect on the same target cells, such as potential motility through different mechanisms underlying the basic expressive activity of anti-oncogenes like nm23-H1.3. The original formula composed of the same therapeutic principles of Qi-boosting toxin-resolving can obviously inhibit the expressive level of c-myc, the main responsible gene for cellular potential motility, while the revised formula can promote its expression in the target cells CNE2Z. These results indicate that different formula composing methods can induce largely different treating response to the target cells CNE2Z cell even though based on a same therapeutic principle, such as cellular potential motility, through different effect on oncogene nm23-H1. principles of prescription formation inhibit4. The original formula composed of the same therapeutic principle of Qi-boosting toxin-resolving can obviously inhibit the expressive activities of the main cytoskeletal proteins likeβ-tublin, F-actin and ezrin in the target cells, while the revised formula can significantly promote their expression in the target cells. These results indicate that different formula composing methods can induce very different resulting events, like inhibitory effect, or promoting effect, on the potential motility of the same target cells CNE2Z through different affecting mechanisms on responsible genes such as oncogene nm23-H1, even based on the same principle of therapy.5. As a whole, the original formula composed of on the same therapeutic principle of Qi-boosting toxin-resolving can significantly inhibit the biological activities of responsible genes as Rac-1 and p-JNK associated with the bio-mechanic mechanisms in the signaling networks to induce inhibitory effect on cellular motility. In the contrast, the revised formula can obviously promote their biological activities in the same targeting cells.6. To summarize it up, the original formula, composed of the same therapeutic principle of Qi-boosting toxin-resolving, may play a suppressing effect on cellular potential motility. On the contrast, the revised formula can promote this kind of cellular biological property. These results indicate that different formula composing technique, even though based on the same therapeutic principle, can induce completely different pharmacological effects on the same kind of targets. This means that the formula composing methods, even based on a same treating principle, may give rise very important significance for clinical practice.
|
PDF Full Text Request